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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.

A hydrodynamic-based microfluidic chip consisted of two function units that could not only separate tumor cells (TCs) from whole blood but also remove residual blood cells was designed. The separation of TCs was achieved by a straight contraction-expansion array (CEA) microchannel on the front end of the chip. The addition of contractive structure brought a micro-vortex like Dean vortex that promoted cell focusing in the channel, while when cells entered the dilated region, the wall-induced lift force generated by the channel wall gave cells a push away from the wall. As the wall-induced lift force is proportional to the third power of the cell diameter, TCs with larger diameter will have a larger lateral migration under the wall-induced lift force, realizing the separation of TCs from blood sample. Fluorescent particles with diameters of 19.3 μm and 4.5 μm were used to simulate TCs and red blood cells, respectively, to verify the separation capacity of the proposed CEA microchannel for particles with different diameter. And a separation efficiency 98.7% for 19.3 μm particles and a removal rate 96.2% for 4.5 μm particles was observed at sample flow rate of 10 μL min -1 and sheath flow rate of 190 μL min -1 . In addition, a separation efficiency about 96.1% for MCF-7 cells (stained with DiI) and removal rates of 96.2% for red blood cells (RBCs) and 98.7% for white blood cells (WBCs) were also obtained under the same condition. However, on account of the large number of blood cells in the blood, there will be a large number of blood cells remained in the isolated TCs, so a purification unit based on hydrodynamic filtration (HDF) was added after the separation microchannel. The purification channel is a size-dictated cell filter that can remove residual blood cells but retain TCs, thus achieving the purification of TCs. Combined the CEA microchannel and the purifier, the microchip facilitates sorting of MCF-7 cells from whole blood with a separation rate about 95.3% and a removal rate over 99.99% for blood cells at a sample flow rate of 10 μL min -1 , sheath flow rate of 190 μL min -1 and washing flow rate of 63 μL min -1 ., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Bioanalysis faces challenges in achieving fast, reliable, and point-of-care (POC) determination methods for timely diagnosis and prognosis of diseases. POC devices often display lower sensitivity compared to laboratory-based methods, limiting their ability to quantify low concentrations of target analytes. To enhance sensitivity, the synthesis of new materials and improvement of the efficiency of the analytical strategies are necessary. Enzyme-mimicking materials have revolutionized the field of the fabrication of new high-throughput sensing devices. The integration of microfluidic chips with analytical techniques offers several benefits, such as easy miniaturization, need for low biological sample volume, etc., while also enhancing the sensitivity of the probe. The use enzyme-like nanomaterials in microfluidic systems can offer portable strategies for real-time and reliable detection of biological agents. Colorimetry and electrochemical methods are commonly utilized in the fabrication of nanozyme-based microfluidic systems. The review summarizes recent developments in enzyme-mimicking materials-integrated microfluidic analytical methods in biomedical analysis and discusses the current challenges, advantages, and potential future directions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

High demands for food safety detection and analysis have been advocated with people's increasing living standards. Even though numerous analytical testing techniques have been proposed, their widespread adoption is still constrained by the high limit of detection, narrow detection ranges, and high implementation costs. Due to their advantages, such as reduced sample and reagent consumption, high sensitivity, automation, low cost, and portability, using microfluidic devices for food safety monitoring has generated significant interest. This review provides a comprehensive overview of the latest microfluidic detection platforms (published in recent 4 years) and their applications in food safety, aiming to provide references for developing efficient research strategies for food contaminant detection and facilitating the transition of these platforms from laboratory research to practical field use., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Microfluidic systems find widespread applications in diagnostics, biological research, chemistry, and engineering studies. Among their many critical parameters, flow rate plays a pivotal role in maintaining the functionality of microfluidic systems, including droplet-based microfluidic devices and those used in cell culture. It also significantly influences microfluidic mixing processes. Although various flow rate measurement devices have been developed, the challenge remains in accurately measuring flow rates within customized channels. This paper presents the development of a 3D-printed smartphone-based flow velocity meter. The 3D-printed platform is angled at 30° to achieve transparent flow visualization, and it doesn't require any external optical components such as external lenses and filters. Two LED modules integrated into the platform create a uniform illumination environment for video capture, powered directly by the smartphone. The performance of our platform, combined with a customized video processing algorithm, was assessed in three different channel types: uniform straight channels, straight channels with varying widths, and vessel-like channel patterns to demonstrate its versatility. Our device effectively measured flow velocities from 5.43 mm/s to 24.47 mm/s, with video quality at 1080p resolution and 60 frames per second, for which the measurement range can be extended by adjusting the frame rate. This flow velocity meter can be a useful analytical tool to evaluate and enhance microfluidic channel designs of various lab-on-a-chip applications., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Orientationally immobilized enzyme microreactors (OIMERs), embedded in microfluidic paper-based analytical devices (μPADs) were developed for improved detection of pesticide residues in food. Acetylcholinesterase (AChE) was orientationally immobilized on the reusable Part I of the μPADs, using the specific affinity binding of concanavalin A (Con A) to a glycosyl group on AChE. Using the disposable Part II, facile colorimetric quantification was performed with a smartphone and software, or qualitative detection by a naked-eye visual test. The AChE immobilized in OIMERs not only had improved activity and stability, but also high sensitivity, with a limit of detection as low as (0.007 ± 0.003) μg/mL. The method was used to detect pesticides residues in real vegetable samples; the recovery (88.6-102.7%) showed high reliability for pesticide residues detection in foods. A molecular docking study and an enzyme kinetic analysis were conducted to characterize the mechanism of action of the OIMERs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Metastatic tumors have poor prognoses for progression-free and overall survival for all cancer patients. Rare circulating tumor cells (CTCs) and rarer circulating tumor cell clusters (CTCCs) are potential biomarkers of metastatic growth, with CTCCs representing an increased risk factor for metastasis. Current detection platforms are optimized for ex vivo detection of CTCs only. Microfluidic chips and size exclusion methods have been proposed for CTCC detection; however, they lack in vivo utility and real-time monitoring capability. Confocal backscatter and fluorescence flow cytometry (BSFC) has been used for label-free detection of CTCCs in whole blood based on machine learning (ML) enabled peak classification. Here, we expand to a deep-learning (DL)-based, peak detection and classification model to detect CTCCs in whole blood data. We demonstrate that DL-based BSFC has a low false alarm rate of 0.78 events per min with a high Pearson correlation coefficient of 0.943 between detected events and expected events. DL-based BSFC of whole blood maintains a detection purity of 72% and a sensitivity of 35.3% for both homotypic and heterotypic CTCCs starting at a minimum size of two cells. We also demonstrate through artificial spiking studies that DL-based BSFC is sensitive to changes in the number of CTCCs present in the samples and does not add variability in detection beyond the expected variability from Poisson statistics. The performance established by DL-based BSFC motivates its use for in vivo detection of CTCCs. Using transfer learning, we additionally validate DL-based BSFC on blood samples from different species and cancer cell types. Further developments of label-free BSFC to enhance throughput could lead to critical applications in the clinical detection of CTCCs and ex vivo isolation of CTCC from whole blood with minimal disruption and processing steps.

Concentration gradient generation and mixed combinations of multiple solutions are of great value in the field of biomedical research. However, existing concentration gradient generators for single or two-drug solutions cannot simultaneously achieve multiple concentration gradient formations and mixed solution combinations. Furthermore, the whole system was huge, and required expensive auxiliary equipment, which may lead to complex operations. To address this problem, we devised a novel 3D microchannel network design, which is capable of creating all the desired mixture combinations and concentration gradients of given small amounts of the input solutions. As a proof of concept, the device we presented was verified by both colorimetric and fluorescence detection methods to test the efficiency. This can enable the implementation of one to three solutions with no driving pump and facilitate unique multiple types of more concentration gradients and mixture combinations in a single operation. We envision that this will be a promising candidate for the development of simplified methods for screening of the appropriate concentration and combination, such as various drug screening applications.

Microfluidics technology is promising in developing microparticle manipulation technology due to its nondestructive control and notable adaptability. The manipulation of microparticle based on swirling stagnation point is one of the feasible microfluidics biotechnologies. Aiming to improve the regulation and control of microparticle, baffle wall is introduced into the 2-microchannel flow field. The theory of wall attachment jet is employed to elucidate the effect of baffle wall. Subsequently, finite volume method simulation is conducted by modeling the swirling flow region (SFR), and the swirling strength is calculated to characterize the SFR's particle-capturing ability. Experimental validation of the modeling and simulation methods is performed using a printed microfluidic chip, which has demonstrated exceptional reliability. Simulation results show that the baffle wall makes considerable influence on the SFR. Strikingly, a global range adjustment of stagnation point is realized when the baffle wall is configured with a convex shape, which has remarkably outperformed our previous work, where the stagnation point could only move within half range of the field. This work significantly contributes to advanced flow field structure and provides insight into better regulation of stagnation point as well as microparticles. These findings have potential applications in the analysis of the effect of bio/chemical substances on single cell., (© 2023 International Union of Biochemistry and Molecular Biology, Inc.)

The challenges in the management of human diseases are largely determined by the precision, speed and ease of diagnostic procedures available. Developments in biomedical engineering technologies have greatly helped in transforming human health care, especially for disease diagnosis which in turn lead to better patient outcomes. One such development is in the form of microfluidic chip technology which has transformed various aspects of human health care. We present in this review, a comprehensive account on the utility of microfluidic chip technologies for the diagnosis of autoimmune disorders, cardiovascular diseases (CVDs), infectious diseases, and neurodegenerative conditions. We have included the diseases posing global threat such as rheumatoid arthritis, diabetes, pernicious anemia, tuberculosis, COVID-19, influenza, alzheimer's, multiple sclerosis, and epilepsy. Apart from discussing the ways of microfluidic chip in diagnosis, we included a section presenting electrochemical, electrical, optical, and acoustic detection technologies for the precise diagnosis of CVDs. Microfluidics platforms have thus revolutionized novel capabilities in addressing the requirements of point-of-care diagnostics enabling miniaturization by integrating multiple laboratory functions into a single chip resulting in "one flow - one solution" systems. Hence, the precision and early diagnoses of diseases are now possible due to the advancements of microfluidics-based technology., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

A common challenge in microfluidic cell cultures has to do with analysis of cell function without replacing a significant fraction of the culture volume and disturbing local concentration gradients of signals. To address this challenge, we developed a microfluidic cell culture device with an integrated bioanalysis unit to enable on-chip analysis of picoliter volumes of cell-conditioned media. The culture module consisted of an array of 140 microwells with a diameter of 300 m which were made low-binding to promote organization of cells into 3D spheroids. The bioanalysis module contained a droplet generator unit, 15 micromechanical valves and reservoirs loaded with reagents. Each 0.8 nL droplet contained an aliquot of conditioned media mixed with assay reagents. The use of microvalves allowed us to load enzymatic assay and immunoassay into sequentially generated droplets for detection of glucose and albumin, respectively. As a biological application of the microfluidic device, we evaluated hormonal stimulation and glucose consumption of hepatic spheroids. To mimic physiological processes occurring during feeding and fasting, hepatic spheroids were exposed to pancreatic hormones, insulin or glucagon. The droplet-based bioanalysis module was used to measure uptake or release of glucose upon hormonal stimulation. In the future, we intend to use this microfluidic device to mimic and measure pathophysiological processes associated with hepatic insulin resistance and diabetes in the context of metabolic syndrome., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Infections, autoimmune diseases, desired and adverse immunological responses to treatment can lead to a complex and dynamic cytokine response in vivo. This response involves numerous immune cells secreting various cytokines to orchestrate the immune reaction. However, the secretion dynamics, amounts, and co-occurrence of the different cytokines by various cell subtypes remain poorly understood due to a lack of appropriate tools to study them. Here, we describe a protocol using a microfluidic droplet platform that allows the time-resolved quantitative measurement of secretion dynamics for several cytokines in parallel on the single-cell level. This is enabled by the encapsulation of individual cells into microfluidic droplets together with a multiplexed immunoassay for parallel quantification of cytokine concentrations, their immobilization for dynamic fluorescent imaging, and the analysis of the respective images to derive secreted quantities and dynamics. The protocol describes the preparation of functionalized magnetic nanoparticles, calibration experiments, cell preparation, and the encapsulation of the cells and nanoparticles into droplets for fluorescent imaging and subsequent image and data analysis using the example of lipopolysaccharide-stimulated human peripheral blood mononuclear cells. The presented platform identified distinct cytokine secretion behavior for single and co-secreting cells, characterizing the expected phenotypic heterogeneity in the measured cell sample. Furthermore, the modular nature of the assay allows its adaptation and application to study a variety of proteins, cytokines, and cell samples, potentially leading to a deeper understanding of the interplay between different immune cell types and the role of the different cytokines secreted dynamically to shape the tightly regulated immune response. These new insights could be particularly interesting in the studies of immune dysregulations or in identifying target populations in therapy and drug development.

The gold standard for nucleic acid amplification-based diagnosis is the polymerase chain reaction (PCR). The PCR recognizes the targets such as foodborne pathogens by amplifying their specific genes. The integration of nucleic acid amplification-based assays on microfluidic platforms represents a highly promising solution for convenient, cheap, and effective control of foodborne pathogens. However, the application of the PCR is limited to on-site detection because the method requires sophisticated equipment for temperature control, which makes it complicated for microfluidic integration. Alternatively, isothermal amplification methods are promising tools for integrating microfluidic platforms for on-site detection of foodborne pathogens. This review summarized advances in isothermal amplification-based microfluidic devices for detecting foodborne pathogens. Different nucleic acid extraction approaches and the integration of these approaches in microfluidic platforms were first reviewed. Microfluidic platforms integrated with three common isothermal amplification methods including loop-mediated isothermal amplification, recombinase polymerase amplification, and recombinase-aided amplification were then described and discussed.

Numerous applications in medical diagnostics, cell engineering therapy, and biotechnology require the identification and sorting of cells that express desired molecular surface markers. We developed a microfluidic method for high-throughput and label-free sorting of biological cells by their affinity of molecular surface markers to target ligands. Our approach consists of a microfluidic channel decorated with periodic skewed ridges and coated with adhesive molecules. The periodic ridges form gaps with the opposing channel wall that are smaller than the cell diameter, thereby ensuring cell contact with the adhesive surfaces. Using three-dimensional computer simulations, we examine trajectories of adhesive cells in the ridged microchannels. The simulations reveal that cell trajectories are sensitive to the cell adhesion strength. Thus, the differential cell trajectories can be leveraged for adhesion-based cell separation. We probe the effect of cell elasticity on the adhesion-based sorting and show that cell elasticity can be utilized to enhance the resolution of the sorting. Furthermore, we investigate how the microchannel ridge angle can be tuned to achieve an efficient adhesion-based sorting of cells with different compliance.

The use of microfluidic paper-based analytical devices (μPADs) for aiding medical diagnosis is a growing trend in the literature mainly due to their low cost, easy use, simple manufacturing, and great potential for application in low-resource settings. Many important biomarkers (proteins, ions, lipids, hormones, DNA, RNA, drugs, whole cells, and more) and biofluids are available for precise detection and diagnosis. We have reviewed the advances μPADs in medical diagnostics have achieved in the last few years, focusing on the most common human biofluids (whole blood/plasma, sweat, urine, tears, and saliva). The challenges of detecting specific biomarkers in each sample are discussed, along with innovative techniques that overcome such limitations. Finally, the difficulties of commercializing μPADs are considered, and future trends are presented, including wearable devices and integrating multiple steps in a single platform., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Separation of malignant tumor cells (MTCs) from large background cells in untreated malignant pleural and peritoneal effusions (MPPEs) is critical for improving the sensitivity and efficiency of cytological diagnosis. Herein, we proposed a cascaded elasto-inertial cell separation (CEICS) device integrating an interfacial elasto-inertial microfluidic channel with a symmetric contraction expansion array (CEA) channel for pretreatment-free, high-recovery-ratio, and high-purity separation of MTCs from clinical MPPEs. First, the effects of flow-rate ratio, cell concentration, and cell size on separation performances in two single-stage channels were investigated. Then, the performances of the integrated CEICS device were characterized using blood cells spiked with three different tumor cells (MCF-7, MDA-MB-231, and A549 cells) at a high total throughput of 240 μL min -1 . An average recovery ratio of ∼95% and an average purity of ∼61% for the three tumor cells were achieved. Finally, we successfully applied the CEICS device for the pretreatment-free separation of MTCs from clinical MPPEs of different cancers. Our CEICS device may provide a preparation tool for improving the sensitivity and efficiency of cytological examination.

Nanostructure-enhanced biodetection is widely used for early diagnosis and treatment, which plays an essential role in improving the cure rates of cancer patients. ZnO nanostructure-based fluorescence immunoassay has been demonstrated to enable effective and sensitive detection of cancer biomarkers for their excellent biocompatibility, high electrical point, and unique fluorescence enhancement properties. Further optimization of such fluorescence detection technology is still in demand to meet the requirements of highly sensitive, multiplex detection, and user-friendly devices. Droplet microfluidics is a promising platform for high-throughput analysis of biological assays, and they have been intensively used in analytical chemistry and synthesis of nanoparticles. Here, we propose a simple droplet chip, where a static droplet array was successfully obtained for in situ growth of ZnO nanostructures with varied diameters by changing the entire growth time and replenishment interval. This device provides a novel and alternative approach for patterned growth of ZnO nanostructures and understanding the growth condition of ZnO nanostructures in static droplet, which offers some guidance toward the design of multiple fluorescence amplification platforms potentially for biosensing. As a demonstration, we used the patterned grown ZnO nanostructures for multiple detection of cancer biomarkers, achieving a low limit of detection as low as 138 fg/mL in the human α-fetoprotein assay and 218 fg/mL in the carcinoembryonic antigen assay with a large dynamic range of 8 orders. These results suggest that such multifunctional microfluidic devices may be useful tools for efficient fluorescence diagnostic assays.

This study presents a novel miniaturized device as a 3D-printed microfluidic magnetic platform specifically designed to manipulate magnetic microparticles in a microfluidic chip for rapid deoxyribonucleic acid (DNA) isolation. The novel design enables the movement of the magnetic particles in the same or opposite directions with the flow or suspends them in continuous flow. A computational model was developed to assess the effectiveness of the magnetic manipulation of the particles. Superparamagnetic monodisperse silica particles synthesized in-house are utilized for the isolation of fish sperm DNA and human placenta DNA. It was demonstrated that the proposed platform can perform DNA isolation within 10 min with an isolation efficiency of 50% at optimum operating conditions.

Iodine is an essential micronutrient for humans due to its fundamental role in the biosynthesis of thyroid hormones. As a key parameter to assess health conditions, iodine intake needs to be monitored to ascertain and prevent iodine deficiency. Iodine is available from various food sources (such as seaweed, fish, and seafood, among others) and dietary supplements (multivitamins or mineral supplements). In this work, a microfluidic paper-based analytical device (μPAD) to quantify iodide in seaweed and dietary supplements is described. The developed μPAD is a small microfluidic device that emerges as quite relevant in terms of its analytical capacity. The quantification of iodide is based on the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by hydrogen peroxide in the presence of iodine, which acts as the catalyst to produce the blue form of TMB. Additionally, powder silica was used to intensify and uniformize the colour of the obtained product. Following optimization, the developed μPAD enabled iodide quantification within the range of 10-100 µM, with a detection limit of 3 µM, and was successfully applied to seaweeds and dietary supplements. The device represents a valuable tool for point-of-care analysis, can be used by untrained personnel at home, and is easily disposable, low-cost, and user-friendly.

The application of microfluidic devices as next-generation cell and tissue culture systems has increased impressively in the last decades. With that, a plethora of materials as well as fabrication methods for these devices have emerged. Here, we describe the rapid prototyping of microfluidic devices, using micromilling and vapour-assisted thermal bonding of polymethyl methacrylate (PMMA), to create a spheroid-on-a-chip culture system. Surface roughness of the micromilled structures was assessed using scanning electron microscopy (SEM) and atomic force microscopy (AFM), showing that the fabrication procedure can impact the surface quality of micromilled substrates with milling tracks that can be readily observed in micromilled channels. A roughness of approximately 153 nm was created. Chloroform vapour-assisted bonding was used for simultaneous surface smoothing and bonding. A 30-s treatment with chloroform-vapour was able to reduce the surface roughness and smooth it to approximately 39 nm roughness. Subsequent bonding of multilayer PMMA-based microfluidic chips created a durable assembly, as shown by tensile testing. MDA-MB-231 breast cancer cells were cultured as multicellular tumour spheroids in the device and their characteristics evaluated using immunofluorescence staining. Spheroids could be successfully maintained for at least three weeks. They consisted of a characteristic hypoxic core, along with expression of the quiescence marker, p27 kip1 . This core was surrounded by a ring of Ki67-positive, proliferative cells. Overall, the method described represents a versatile approach to generate microfluidic devices compatible with biological applications., (© 2024. The Author(s).)

Background: Nucleic acid extraction (NAE) is an essential step in the whole process of nucleic acid detection (NAT). Traditional manual extraction methods are time-consuming and laborious, unfavorable to the point-of-care testing of nucleic acids. Ultrasound has been emphasized due to its noncontact and easy-to-manipulate characteristics, and integration with microfluidic chip can realize rapid NAE through acoustic streaming effect. The uniformity of magnetic bead mixing in this process is a critical factor affecting the extraction effect. In this study, we developed an ultrasound-assisted NAE technique based on the magnetic bead method and optimized the chip structure to achieve rapid NAE., Result: We use ultrasonic-assisted coupled with magnetic bead method for ultra-fast NAE. The mixing process of magnetic beads driven by acoustic streaming is simulated by a dispersive two-phase flow model, and the ultrasonic incidence angle (θ in ), cone structure aspect ratio (D c /H c ) and sheet structure thickness (H p ) are optimized to enhance the mixing performance. Furthermore, the effectiveness of NAE is validated by utilizing quantitative real-time PCR (qPCR) detection. The findings reveal that a θ in value of 10° yields superior mixing performance compared to other incidence angles, resulting in a maximum increase of 84 % in mixing intensity. When D c /H c  = 0.5 and H p  = 0.5 mm, the maximum mixing index in the localized region of the chamber after 1 s of ultrasound action can reach 83.6 % and 92.5 %, respectively. Compared to the original chamber, the CT values extracted after 5 s of ultrasound action shifted forward by up to 1.9 ct and 4.1 ct, respectively., Significance: The dispersed two-phase flow model can effectively simulate the mixing process of magnetic beads, which plays an important role in assisting the structural design of chip extraction chambers. The single-step mixing of ultrasound-assisted NAE takes only 15s to achieve an extraction performance comparable to manual extraction. The extraction process can be completed within 7 min after integrating this technology with microfluidic chips and automated equipment, providing a solution for automated and efficient NAE., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Previous on-chip technologies for characterizing the cellular mechanical properties often suffer from a low throughput and limited sensitivity. Herein, an inertial multi-force deformability cytometry (IMFDC) is developed for high-throughput, high-accuracy, and high-applicability tumor cell mechanotyping. Three different deformations, including shear deformations and stretch deformations under different forces, are integrated with the IMFDC. The 3D inertial focusing of cells enables the cells to deform by an identical fluid flow, and 10 parameters, such as cell area, perimeter, deformability, roundness, and rectangle deformability, are obtained in three deformations. The IMFDC is able to evaluate the deformability of different cells that are sensitive to different forces on a single chip, demonstrating the high applicability of the IMFDC in analyzing different cell lines. In identifying cell types, the three deformations exhibit different mechanical responses to cells with different sizes and deformability. A discrimination accuracy of ≈93% for both MDA-MB-231 and MCF-10A cells and a throughput of ≈500 cells s -1 can be achieved using the multiple-parameters-based machine learning model. Finally, the mechanical properties of metastatic tumor cells in pleural and peritoneal effusions are characterized, enabling the practical application of the IMFDC in clinical cancer diagnosis., (© 2023 Wiley-VCH GmbH.)

Centrifugal microfluidics holds the potential to revolutionize point-of-care (POC) testing by simplifying laboratory tests through automating fluid and cell manipulation within microfluidic channels. This technology can facilitate blood testing, the most frequent clinical test, at the POC. However, an integrated centrifugal microfluidic device for complete blood counting (CBC) has not yet been fully realized. To address this, we propose an integrated portable system comprising a centrifuge and a hybrid microfluidic disc specifically designed for CBC analysis at the POC. The disc enables the implementation of various spin profiles in different stages of CBC to facilitate in-situ cell separation, solution metering and mixing, and differential cell counting. Furthermore, our system is coupled with a custom script that automates the process and ensures precise quantification of cells using light and fluorescent images captured from the detection chamber of the disc. We demonstrate a close correlation between the proposed method and the hematology analyzer, considered the gold standard, for quantifying hematocrit (R 2  = 0.99), white blood cell count (R 2  = 0.98), white blood cell differential count (granulocyte/agranulocyte; R 2  = 0.89), red blood cell count (R 2  = 0.97), and mean corpuscular volume (R 2  = 0.94). The integration of our portable system offers significant advantages, enabling more accessible and affordable CBC testing at the POC. Considering the simplicity, affordability (∼$250 capital cost and <$2 operational cost per test), as well as low power consumption (>100 tests using a typical 24 V/10 Ah battery), this system has the potential to enhance healthcare delivery, particularly in resource-limited settings and remote areas where access to traditional laboratory facilities is limited., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Inertial microfluidics has demonstrated its ability to focus particles in a passive and straightforward manner. However, achieving flow-rate- and particle-size-insensitive focusing in large-dimension channels with a simple design remains challenging. In this study, we developed a spiral microfluidic with a large-dimension channel to achieve inertial focusing. By designing a unique "big buffering area" and a "small buffering area" in the spiral microchannel, we observed the stabilization and acceleration of secondary flow. Our optimized design allowed for efficient (>99.9%) focusing of 15 μm particles within a wide range of flow rates (0.5-4.5 mL/min) during a long operation duration (0-60 min). Additionally, we achieved effective (>95%) focusing of different-sized particles (7, 10, 15, and 30 μm) and three types of tumor cells (K562, HeLa, and MCF-7) near the inner wall of the 1 mm wide outlet when applying different flow rates (1-3 mL/min). Finally, successful 3D cell focusing was achieved within an optimized device, with the cells positioned at a distance of 50 μm from the wall. Our strategy of stabilizing and accelerating Dean-like secondary flow through the unique configuration of a "big buffering area" and a "small buffering area" proved to be highly effective in achieving inertial focusing that is insensitive to the flow rate and particle size, particularly in large-dimension channels. Consequently, it shows great potential for use in hand-operated microfluidic tools for flow cytometry.

We report the screening of angiotensin converting enzyme (ACE) inhibitors on an origami microfluidic paper-based analytical device (μPAD) using colorimetric detection. The hydrolysis product reacts with ninhydrin, resulting in a purple color at the detection zones. Images of the μPADs are captured using a common cell phone and analyzed with Photoshop software. This platform allows six independent colorimetric reactions to take place simultaneously, and the IC 50 values can be obtained in a single run within 22 min. The relative standard deviations of inhibition efficiencies are generally lower than 4.0 % (n = 5). The IC 50 values of captopril and five products from natural plants were obtained and corresponded well with UV methods. The relative deviations between the two methods are within the range of -5 % to +5 %. This work is a proof-of-concept successfully demonstrating the use of μPADs technology to screen enzyme inhibitors from natural products., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Surface charge is a critical feature of microbes that affects their interactions with other cells and their environment. Because bacterial surface charge is difficult to measure directly, it is typically indirectly inferred through zeta potential measurements. Existing tools to perform such characterization are either costly and ill-suited for non-spherical samples or rely on microfluidic techniques requiring expensive fabrication equipment or specialized facilities. Here, we report the application of commercially available PMMA microfluidic chips and open-source data analysis workflows for facile electrokinetic characterization of particles and cells after prior zeta potential measurement with a Zetasizer for calibration. Our workflows eliminate the need for microchannel fabrication, increase measurement reproducibility, and make zeta potential measurements more accessible. This novel methodology was tested with functionalized 1 μm and 2 μm polystyrene beads as well as Escherichia coli MG1655 strain. Measured zeta potentials for these samples were in agreement with literature values obtained by conventional measurement methods. Taken together, our data demonstrate the power of this workflow to broadly enable critical measurements of particle and bacterial zeta potential for numerous applications.

Microfluidic-based analyses of single-cell dynamics in response to dynamic biochemical signals are emerging as pivotal approaches for investigating the effects of extracellular microenvironmental biochemical factors on cellular structure, function, and behavior. However, current devices often fail to consistently apply identical dynamic biochemical signals to trapped cells. In this study, we introduce a novel radially distributed single-cell trapping microfluidic array, designed to quantitatively and consistently apply identical biochemical stimulating signals to each trapped cell. Numerical simulations were employed to optimize microchannel geometry, enhancing trapping efficiency while minimizing signal distortion. Experimental validation demonstrated the trapping success rate and the single-cell trapping efficiency exceeding 99% and 85%, respectively. The microarray's capability to deliver identical dynamic biochemical stimulating signals, with various waveforms, to each unit was confirmed through fluorescein transport tests. Furthermore, we examined the intracellular calcium dynamics of U-2 OS human osteosarcoma cells in response to dynamic ATP signals, observing both single-peak calcium responses and calcium oscillations, which were modelled by a second-order system with a natural frequency of 1.6 mHz. Overall, our proposed microfluidic array offers a robust and valuable framework for advancing the understanding of single-cell dynamics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Digital polymerase chain reaction (dPCR) is an approach for absolute nucleic acid quantification with high sensitivity. Although several successful commercial dPCR devices have been developed to date, further miniaturizing device dimensions, decreasing cross-contamination, and improving automation level are still research highlights. In this study, we developed a fully contamination-free dPCR detection chip with fluorescence flow cytometry and micro droplet approach. A bifunctional cross-structure (BFCS) was designed to realize monodisperse sample droplet generation in forward flow and droplet detection in backward flow with simple pneumatic control and fixed chip position. In order to improve droplet detection efficiency and accuracy, droplets morphology and sequence pattern during microfluidic droplet generation and backward flow droplet detection at the same cross-structure were observed and analyzed under different pneumatic pressures. In addition, during backward flow droplet detection, an optimized declination angle of the chip was applied to increase droplet reflux rates. For the validation of PCR performance, temperature changing processes during PCR cycles were achieved by heating the monodispersed droplet array with a customized PCR amplification device. The fluorescence signal of each droplet right after passing the cross-structure was excitated and detected. The absolute quantification ability of our integrated dPCR microfluidic chip utilizing flow fluorescence cytometry was tested and verified with Influenza A virus gene (from 7.5 copies/μL to 30000 copies/μL). Thus, our platform provides a novel and integrated approach for ddPCR analysis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Single-cell multiplexed phenotypic analysis expands the biomarkers for diagnosis, heralding a new era of precision medicine. Cell secretions are the primary measures of immune function, but single-cell screening remains challenging. Here, a novel cell membrane-based assay was developed using cholesterol-linked antibodies (CLAbs), integrating immunosorbent assays and droplet microfluidics to develop a flexible high-throughput single-cell secretion assay for multiplexed phenotyping. CLAb-grafted single cells were encapsulated in water-in-oil droplets to capture their own secretions. Subsequently, the cells were extracted from droplets for fluorescence labeling and screening. Multiple secretions and surface proteins were simultaneously measured from single cells by flow cytometry. To validate the approach, THP-1 cells, THP-1-derived M1 macrophages, and dendritic cells were assayed, indicating the differentiation efficiency of THP-1 cells under different chemical stimulations. Moreover, peripheral blood mononuclear cells from healthy donors under various stimuli showed varied active immune cell populations (6.62-47.14%). The peripheral blood mononuclear cells (PBMCs) of nasopharyngeal carcinoma patients were analyzed to identify a higher percentage of actively cytokine-secreted single cells in the basal state (2.82 ± 1.48%), compared with that in the health donors (0.70 ± 0.29%).

Circulating tumor cells (CTCs) are tumor cells that spontaneously detach from the primary focus, and early detection and characterization of CTCs is vital for cancer diagnosis and appropriate treatment. Current methods commonly use EpCAM to capture CTCs, but this results in a loss of information on other CTC subsets (EpCAM-negative cells) due to the heterogeneity of CTCs. Here, we report a novel microfluidic device that integrates the capture and release of heterogeneous CTCs directly from whole blood. A spiral chip was designed for the separation of differently sized cells, and larger CTCs were effectively separated from smaller blood cells with a 98% recovery rate. CD146-containing magnetic beads were used to complement the EpCAM-based CTC capture methods, and the capture efficiency of Fe 3 O 4 @Gelatin@CD146/EpCAM increased by 20% over Fe 3 O 4 @Gelatin@EpCAM. Finally, MMP-9 was employed to release CTCs with high efficiency and less damage by degrading gelatins on the surface of Fe 3 O 4 . The established method was successfully applied to CTC capture and release in a simulated patient's whole blood. The developed method achieved enhanced capture and high activity release of heterogeneous CTCs with less interference by blood cells, which contributes to the early detection and clinical downstream analysis of CTCs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Recent advancements in the profiling of proteomes at the single-cell level necessitate the development of quantitative and versatile platforms, particularly for analyzing rare cells like circulating tumor cells (CTCs). In this chapter, we present an integrated microfluidic chip that utilizes magnetic nanoparticles to capture single tumor cells with exceptional efficiency. This chip enables on-chip incubation and facilitates in situ analysis of cell-surface protein expression. By combining phage-based barcoding with next-generation sequencing technology, we successfully monitored changes in the expression of multiple surface markers induced by CTC adherence. This innovative platform holds significant potential for comprehensive screening of multiple surface antigens simultaneously in rare cells, offering single-cell resolution. Consequently, it will contribute valuable insights into biological heterogeneity and human disease., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Nucleic acid detection is an important tool for clinical diagnosis. The purification of the sample is the most time-consuming step in the nucleic acid testing process and will affect the results of the assay. Here, we developed a surface modification-based nucleic acid purification method and designed an accompanying set of centrifugation equipment and chips to integrate the steps of nucleic acid purification on a single platform. The results of experiments with HeLa cells and HPV type 16 as samples showed that the mentioned method had good nucleic acid purification capability and the accompanying equipment greatly simplified the operation of the experimenters in the whole process. Overall, our equipment can improve the efficiency of nucleic acid purification and is suitable for application in larger-scale clinical assays., (© 2024 Wiley-VCH GmbH.)

Paper-based analysis has captivated scientists' attention in the field of analytical chemistry and related areas for the last two decades. Arguably no other area of modern chemical analysis is so broad and diverse in its approaches spanning from simple 'low-tech' low-cost paper-based analytical devices (PADs) requiring no or simple instrumentation, to sophisticated PADs and microfluidic paper-based analytical devices (μPADs) featuring elements of modern material science and nanomaterials affording high selectivity and sensitivity. Correspondingly diverse is the applicability, covering resource-limited scenarios on the one hand and most advanced approaches on the other. Herein we offer a view reflecting this diversity in the approaches and types of devices. The core idea of this article rests in dividing μPADs according to their type into two groups: A) instrumentation-free μPADs for resource-limited scenarios or developing countries and B) instrumentation-based μPADs as futuristic POC devices for e-diagnostics mainly aimed at developed countries. Each of those two groups is presented and discussed with the view of the main requirements in the given area, the most common targets, sample types and suitable detection approaches either implementing high-tech elements or low-tech low-cost approaches. Finally, a socioeconomic perspective is offered in discussing the fabrication and operational costs of μPADs, and, future perspectives are offered., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Whisky adulteration is a prevalent practice driven by the high cost of these beverages. Counterfeiters commonly dilute whisky with less expensive alcoholic beverages, water, food additives, drugs or pharmaceuticals. Paracetamol (PAR), an analgesic drug that mitigates hangovers and headaches, is commonly used to adulterate whisky. Currently, the primary method for quantifying PAR levels is high-performance liquid chromatography, but this technique is both time consuming and usually generates more residues. In this context, the utilization of miniaturized and portable analytical devices becomes imperative for conducting point-of-care/need analyses. These devices offer several advantages, including portability, user-friendliness, low cost, and minimal material wastage. This study proposes the selective distance-based PAR quantification on whisky samples using a paper-based microfluidic analytical device (μPAD). Colorimetric detection on paper-based platforms offers great benefits such as affordability, portability, and the ability to detect PAR without complicated instrumentation. The optimal detection conditions were achieved by introducing 5 μL of a mixture containing 7.5 mmol L -1 of Fe(III) and K 3 [Fe(CN) 6 ] into the detection zone, along with 12 μL of whisky samples into the sample zone. The method exhibited linear behavior within the concentration range from 15 to 120 mg L -1 , with a determination coefficient of 0.998. PAR was quantified in adulterated samples. The results obtained with the paper-based devices were compared with a referenced method, and no significant differences were observed at a confidence level of 95%. The μPAD allowed to determine ca. 1 drop of pharmaceutical medicine PAR of 200 mg mL -1 in 1 L of solution, demonstrating excellent sensitivity. This method offers cost-effective and rapid analysis, reducing the consumption of samples, reagents, and wastes. Consequently, it could be considered a viable and portable alternative for analyzing beverages at criminal scenes, customs, and police operations, thereby enhancing the field of forensics.

Capillary-assisted flow is valuable for utilizing microfluidics-based electrical sensing platforms at on-site locations by simplifying microfluidic operations and system construction; however, incorporating capillary-assisted flow in platforms requires easy microfluidic modification and stability over time for capillary-assisted flow generation and sensing performance. Herein, we report a capillary-assisted microfluidics-based electrical sensing platform using a one-step modification of polydimethylsiloxane (PDMS) with polyethylene glycol (PEG). As a model of electrical sensing platforms, this work focused on resistive pulse sensing (RPS) using a micropore in a microfluidic chip for label-free electrical detection of single analytes, and filling the micropore with an electrolyte is the first step to perform this RPS. The PEG-PDMS surfaces remained hydrophilic after ambient storage for 30 d and assisted in generating an electrolyte flow for filling the micropore with the electrolyte. We demonstrated the successful detection and size analysis of micrometer particles and bacterial cells based on RPS using the microfluidic chip stored in a dry state for 30 d. Combining this capillary-assisted microfluidic platform with a portable RPS system makes on-site detection and analysis of single pathogens possible.

The microfluidic droplet polymerase chain reaction (PCR), which enables simultaneous DNA amplification in numerous droplets, has led to the discovery of various applications that were previously deemed unattainable. Decades ago, it was demonstrated that the temperature holding periods at the denaturation and annealing stages in thermal cycles for PCR amplification could be essentially eliminated if a rapid change of temperature for an entire PCR mixture was achieved. Microfluidic devices facilitating the application of such fast thermocycling protocols have significantly reduced the time required for PCR. However, in microfluidic droplet PCR, ensuring successful amplification from single molecules within droplets has limited studies on accelerating assays through fast thermocycling. Our developed microfluidic cartridge, distinguished for its convenience in executing single-molecule droplet PCR with common laboratory equipment, features droplets positioned on a thin glass slide. We hypothesized that applying fast thermocycling to this cartridge would achieve single-molecule droplet PCR amplification. Indeed, the application of this fast protocol demonstrated successful amplification in just 22 min for 30 cycles (40 s/cycle). This breakthrough is noteworthy for its potential to expedite microfluidic droplet PCR assays, ensuring efficient single-molecule amplification within a remarkably short timeframe.

Significant breakthroughs have been made in digital microfluidic (DMF)-based technologies over the past decades. DMF technology has attracted great interest in bioassays depending on automatic microscale liquid manipulations and complicated multi-step processing. In this review, the recent advances of DMF platforms in the biomedical field were summarized, focusing on the integrated design and applications of the DMF system. Firstly, the electrowetting-on-dielectric principle, fabrication of DMF chips, and commercialization of the DMF system were elaborated. Then, the updated droplets and magnetic beads manipulation strategies with DMF were explored. DMF-based biomedical applications were comprehensively discussed, including automated sample preparation strategies, immunoassays, molecular diagnosis, blood processing/testing, and microbe analysis. Emerging applications such as enzyme activity assessment and DNA storage were also explored. The performance of each bioassay was compared and discussed, providing insight into the novel design and applications of the DMF technology. Finally, the advantages, challenges, and future trends of DMF systems were systematically summarized, demonstrating new perspectives on the extensive applications of DMF in basic research and commercialization., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Flow based deformation cytometry has shown potential for cell classification. We demonstrate the principle with an injection moulded microfluidic chip from which we capture videos of adult and fetal red blood cells, as they are being deformed in a microfluidic chip. Using a deep neural network - SlowFast - that takes the temporal behavior into account, we are able to discriminate between the cells with high accuracy. The accuracy was larger for adult blood cells than for fetal blood cells. However, no significant difference was observed between donors of the two types., (© 2023. The Author(s).)

The counts and phenotypes of circulating tumor cells (CTCs) in whole blood are useful for disease monitoring and prognostic assessment of cancer. However, phenotyping CTCs in the blood is difficult due to the presence of a large number of background blood cells, especially some blood cells with features similar to those of tumor cells. Herein, we presented a viscoelastic-sorting integrated deformability cytometer (VSDC) for high-throughput label-free sorting and high-precision mechanical phenotyping of tumor cells. A sorting chip for removing large background blood cells and a detection chip for detecting multiple cellular mechanical properties were integrated into our VSDC. Our VSDC has a sorting efficiency and a purity of over 95% and over 81% for tumor cells, respectively. Furthermore, multiple mechanical parameters were used to distinguish tumor cells from white blood cells using machine learning. An accuracy of over 97% for identifying tumor cells was successfully achieved with the highest identification accuracy of 99.4% for MCF-7 cells. It is envisioned that our VSDC will open up new avenues for high-throughput and label-free single-cell analysis in various biomedical applications.

Although microfluidic devices have made remarkable strides in blood cell separation, there is still a need for further development and improvement in this area. Herein, we present a novel ultralow aspect ratio ( H / W = 1:36) spiral channel microfluidic device with ordered micro-obstacles for sheathless and flow-rate-insensitive blood cell separation. By introducing ordered micro-obstacles into the spiral microchannels, reduced magnitude fluctuations in secondary flow across different loops can be obtained through geometric confinement. As a result, the unique Dean-like secondary flow can effectively enhance the separation efficiency of particles in different sizes ranging from 3 to 15 μm. Compared to most existing microfluidic devices, our system offers several advantages of easy manufacturing, convenient operation, long-term stability, highly efficient performance (up to 99.70% rejection efficiency, including platelets), and most importantly, insensitivity to cell sizes as well as flow rates (allowing for efficient separation of different-sized blood cells in a wide flow rate from 1.00 to 2.50 mL/min). The unique characteristics, such as ultralow aspect ratio, sequential micro-obstacles, and controlled secondary flow, make our device a promising solution for practical plasma extraction in biomedical research and clinical applications.

Intercellular communication is critical to the understanding of human health and disease progression. However, compared to traditional methods with inefficient analysis, microfluidic co-culture technologies developed for cell-cell communication research can reliably analyze crucial biological processes, such as cell signaling, and monitor dynamic intercellular interactions under reproducible physiological cell co-culture conditions. Moreover, microfluidic-based technologies can achieve precise spatial control of two cell types at the single-cell level with high throughput. Herein, this review focuses on recent advances in microfluidic-based 2D and 3D devices developed to confine two or more heterogeneous cells in the study of intercellular communication and decipher the advantages and limitations of these models in specific cellular research scenarios. This review will stimulate the development of more functionalized microfluidic platforms for biomedical research, inspiring broader interests across various disciplines to better comprehend cell-cell communication and other fields, such as tumor heterogeneity and drug screening., (© 2023 IOP Publishing Ltd.)

Sensitive biomarker detection techniques are beneficial for both disease diagnosis and postoperative examinations. In this study, we report an integrated microfluidic chip designed for the immunodetection of prostate-specific antigens (PSAs). The microfluidic chip is based on the three-dimensional structure of quartz capillaries. The outlet channel extends to 1.8 cm, effectively facilitating the generation of uniform droplets ranging in size from 3 to 50 μm. Furthermore, we successfully immobilized the captured antibodies onto the surface of magnetic beads using an activator, and we constructed an immunosandwich complex by employing biotinylated antibodies. A key feature of this microfluidic chip is its integration of microfluidic droplet technology advantages, such as high-throughput parallelism, enzymatic signal amplification, and small droplet size. This integration results in an exceptionally sensitive PSA detection capability, with the detection limit reduced to 7.00 ± 0.62 pg/mL., (© 2023 Wiley-VCH GmbH.)

Organelle size varies with normal and abnormal cell function. Thus, size-based particle separation techniques are key to assessing the properties of organelle subpopulations differing in size. Recently, insulator-based dielectrophoresis (iDEP) has gained significant interest as a technique to manipulate sub-micrometer-sized particles enabling the assessment of organelle subpopulations. Based on iDEP, we recently reported a ratchet device that successfully demonstrated size-based particle fractionation in combination with continuous flow sample injection. Here, we used a numerical model to optimize the performance with flow rates a factor of three higher than previously and increased the channel volume to improve throughput. We evaluated the amplitude and duration of applied low-frequency DC-biased AC potentials improving separation efficiency. A separation efficiency of nearly 0.99 was achieved with the optimization of key parameters-improved from 0.80 in previous studies (Ortiz et al. Electrophoresis, 2022;43;1283-1296)-demonstrating that fine-tuning the periodical driving forces initiating the ratchet migration under continuous flow conditions can significantly improve the fractionation of organelles of different sizes., (© 2023 Wiley-VCH GmbH.)

A simple, low-cost, three-dimensional (3D) lab-on-a-foil microfluidic device for dielectrophoretic separation of circulating tumor cells (CTCs) is designed and constructed. Disposable thin films are cut by xurography and microelectrode array are made with rapid inkjet printing. The multilayer device design allows the studying of spatial movements of CTCs and red blood cells (RBCs) under dielectrophoresis (DEP). A numerical simulation was performed to find the optimum driving frequency of RBCs and the crossover frequency for CTCs. At the optimum frequency, RBCs were lifted 120 µm in z-axis direction by DEP force, and CTCs were not affected due to negligible DEP force. By utilizing the displacement difference, the separation of CTCs (modeled with A549 lung carcinoma cells) from RBCs in z-axis direction was achieved. With the nonuniform electric field at optimized driving frequency, the RBCs were trapped in the cavities above the microchannel, whereas the A549 cells were separated with a high capture rate of 86.3% ± 0.2%. The device opens not only the possibility for 3D high-throughput cell separation but also for future developments in 3D cell manipulation through rapid and low-cost fabrication., (© 2023 The Authors. Electrophoresis published by Wiley-VCH GmbH.)

Digital PCR (dPCR) enables sensitive and precise quantification of template nucleic acid without calibration. However, dPCR is not yet in widespread use, probably due to the need for expensive specialized instruments. In this paper, we describe a dPCR system using a simple microfluidic chip and common laboratory tools. The microfluidic chip consists of two parts: a PDMS part with 24,840 × 0.25 nL microwells and a PDMS-coated flat glass plate. Human RNase P gene was adopted as the model template. Commercial products of human genomic DNA and real-time PCR reagents were mixed to make a PCR mixture. The PCR mixture was confined to the microwells by the PDMS degas-driven liquid control technique. The thermal cycling was performed on a common well-type thermal cycler with a minor modification. During the thermal cycling, evaporation of the PCR mixture was prevented with a handmade water holder. In the fluorescence image, bright (positive) microwells and dim (negative) ones were clearly discriminated. The number of the positive microwells was counted using software, and was used for estimation of the template concentration in the sample based on the theory of the Poisson distribution. The estimated concentrations well agreed with the input template concentrations in the range from 1.32 copies/µL to 13 200 copies/µL. The techniques presented in this paper will pave the way for facile dPCR in a broad range of laboratories without the need for expensive instruments., (© 2023. The Author(s), under exclusive licence to The Japan Society for Analytical Chemistry.)

The separation of wear microparticles in lubricating oil is crucial for improving the accuracy and throughput of the subsequent detection. However, there are few kinds of research on the separation of high-density metallic microparticles in high-viscosity lubricating oil. In this paper, a passive method for separating the metallic microparticles in oil is proposed. Gravity sedimentation was adopted to realize three-dimensional (3D) focusing of the particle by using an inclined capillary. The gravity-based 3D focusing made the sheath flow no longer responsible for the particle focusing and effectively reduced the sheath flow. Then, the separation of different-sized metallic microparticles was achieved in a horizontal channel with the aid of a sheath flow based on the different driving forces. The present method solved the problem of nonsynchronous separation of the particle in comparison to the traditional methods. This device has a simple structure with high separation efficiency, and it is easy to integrate with the detection channel. The influence of numerous parameters on the gravity-based focusing and separation was systematically studied by the numerical simulation and the experiment. The design criteria were established, which is useful in designing and employing the device, expanding its application to other non-neutral buoyancy particle separation cases, and opening up more prospects for microfluidic technology., (© 2023 Wiley-VCH GmbH.)

The isolation of single cell or droplet is first and crucial step to single-cell analysis, which is important for cancer research and diagnostic methods. This review provides an overview of technologies that are currently used or in development to realize the isolation. Microfluidic based manipulation is an emerging technology with the distinct advantages of miniaturization and low cost. Therefore, recent developments in microfluidic isolated methods have attracted extensive attention. We introduced herein five strategies based on microfluid: trap, microfluidic discrete manipulation, bioprinter, capillary and inertial force. For every technology, their basic principles and features were discussed firstly. Then some modified approaches and applications were listed as the extension. Finally, we compared the advantages and drawbacks of these methods, and analyzed the trend of the manipulation based on microfluidics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Isolation and detection of circulating tumor cells (CTCs) hold significant importance for the early diagnosis of cancer and the assessment of therapeutic strategies. However, the scarcity of CTCs among peripheral blood cells presents a major challenge to their detection. Additionally, a similar size range between CTCs and white blood cells (WBCs) makes conventional microfluidic platforms inadequate for the isolation of CTCs. To overcome these challenges, in this study, a novel inertial-dielectrophoretic microfluidic channel for size-independent, single-stage separation of CTCs from WBCs has been presented. The proposed device utilizes a spiral microchannel embedded with interdigitated electrodes. A numerical model is developed and validated to investigate the influence of various parameters related to the channel design, fluid flow, and electrode configuration. It was found that optimal separation of CTCs could be obtained at a relatively low voltage, termed the critical voltage. Furthermore, at the critical voltage of 7.5 V, the hybrid microchannel is demonstrated to be capable of separating CTCs from different WBC subtypes including granulocytes, monocytes, T-, and B-lymphocytes. The unique capabilities of the hybrid spiral microchannel allow for this size-independent isolation of CTCs from a mixture of WBCs. Overall, the proposed technique can be readily utilized for continuous and high-throughput separation of cancer cells., (© 2023 Wiley-VCH GmbH.)

Pillar shape exploration in deterministic lateral displacement (DLD) technique holds great promise for developing high-performance microfluidic devices with versatile sorting schemes. A recent innovative design using filter-like micropillars was proposed to improve cell separation, but its significance might be greatly underestimated due to an inaccurate understanding of the underlying mechanism. In this study, we employ mesoscopic hydrodynamic simulations to explore the movement and separation of rigid spherical particles in DLD arrays using various two-piece hybrid (TPH) pillars, where each pillar consists of two individual pieces separated by a tunable inter-piece channel. In comparison with the conventional one-piece pillars, the back piece of TPH-pillars is found to hierarchically tailor the flow profile of the front piece on the basis of the row shift fraction and the inter-piece channel width, resulting in unique tunable multi-scheme separation at low, intermediate, and high row shift fractions, respectively. At the intermediate regime, in particular, the first flow lane that determines the critical separation size could be physically fenced out by the inter-piece channel, and a delicate coupling of hydrodynamic filtration and DLD has been revealed to induce a constant critical size in the whole regime. This work theoretically demonstrates the feasibility and significance of TPH-pillars, which may open up a new direction of the geometry design by exploiting rich multi-piece hybrid structures to expand the versatility of the DLD technique., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Microfluidic technologies enabling the control of secondary flow are essential for the successful separation of blood cells, a process that is beneficial for a wide range of medical research and clinical diagnostics. Herein, we introduce a dimension-confined microfluidic device featuring a double-spiral channel designed to regulate secondary flows, thereby enabling high-throughput isolation of blood for plasma extraction. By integrating a sequence of micro-obstacles within the double-spiral microchannels, the stable and enhanced Dean-like secondary flow across each loop can be generated. This setup consequently prompts particles of varying diameters (3, 7, 10, and 15 μm) to form different focusing states. Crucially, this system is capable of effectively separating blood cells of different sizes with a cell throughput of (2.63-3.36) × 10 8 cells/min. The concentration of blood cells in outlet 2 increased 3-fold, from 1.46 × 10 8 to 4.37 × 10 8 , while the number of cells, including platelets, exported from outlets 1 and 3 decreased by a factor of 608. The engineering approach manipulating secondary flow for plasma extraction points to simplicity in fabrication, ease of operation, insensitivity to cell size, high throughput, and separation efficiency, which has potential utility in propelling the development of miniaturized diagnostic devices in the field of biomedical science.