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Heavy metal contamination poses an escalating global challenge to soil ecosystems, with hyperaccumulators playing a crucial role in environmental remediation and resource recovery. The enrichment of diazotrophs and resulting nitrogen accumulation promoted hyperaccumulator growth and facilitated phytoremediation. Nonetheless, the regulatory mechanism of hyperaccumulator biological nitrogen fixation has remained elusive. Here, we report the mechanism by which arsenic regulates biological nitrogen fixation in the arsenic-hyperaccumulator Pteris vittata. Field investigations and greenhouse experiments, based on multi-omics approaches, reveal that elevated arsenic stress induces an enrichment of key diazotrophs, enhances plant nitrogen acquisition, and thus improves plant growth. Metabolomic analysis and microfluidic experiments further demonstrate that the upregulation of specific root metabolites plays a crucial role in recruiting key diazotrophic bacteria. These findings highlight the pivotal role of nitrogen-acquisition mechanisms in the arsenic hyperaccumulation of Pteris vittata, and provide valuable insights into the plant stress resistance. Elevated arsenic is found to enhance plant nitrogen acquisition and plant growth of the arsenic hyperaccumulator Pteris vittate. Multi-omics analysis reveals the interaction between root metabolites and key diazotrophs underlying this effect. [ABSTRACT FROM AUTHOR]

In this study, we developed an instrument-free visual pH determination method using a wedge-shaped microspace with pH-dependent swelling hydrogel particles. This method of pH measurement is achieved by utilizing the property of the hydrogel to swell with increasing pH and the wedge-shaped microspace in a 3D structure. Hydrogel particle formation using a microdroplet-forming chip and an ultraviolet LED enabled the fabrication of spherical pH-responsive hydrogel particles. Microchips with a wedge-shaped microspace of 500–3500 µm could be fabricated using the structures created by a photo-engineered 3D printer as templates. The evaluation of the method using a pH standard demonstrated that the settling distance of the particles decreased with increasing pH, and that the sensitivity and pH resolution were estimated to be 2.94 mm/pH and 0.2 pH, respectively. These results indicated that the method has the potential to visually measure pH. Furthermore, the developed method was applied to the determination of creatinine using creatinine deiminase. The method is readily applicable to biosensing technologies based on pH transduction, making it useful for on-site analysis in a wide range of fields, including medicine, environment, and food. Moreover, it can be adapted to microfluidic analysis for visual quantification using device-free micrototal analysis systems or lab-on-a-chip. [ABSTRACT FROM AUTHOR]

Biofilm formation by Shewanella oneidensis has been extensively studied under oxic conditions; however, relatively little is known about biofilm formation under anoxic conditions and how biofilm architecture and composition can positively influence current generation in bioelectrochemical systems. In this study, we utilized a recently developed microfluidic biofilm analysis setup with automated 3D imaging to investigate the effects of extracellular electron acceptors and synthetic modifications to the extracellular polymeric matrix on biofilm formation. Our results with the wild type strain demonstrate robust biofilm formation even under anoxic conditions when fumarate is used as the electron acceptor. However, this pattern shifts when a graphite electrode is employed as the electron acceptor, resulting in biofilm formation falling below the detection limit of the optical coherence tomography imaging system. To manipulate biofilm formation, we aimed to express BpfG with a single amino acid substitution in the catalytic center (C116S) and to overexpress bpfA. Our analyses indicate that, under oxic conditions, overarching mechanisms predominantly influence biofilm development, rather than the specific mutations we investigated. Under anoxic conditions, the bpfG mutation led to a quantitative increase in biofilm formation, but both strains exhibited significant qualitative changes in biofilm architecture compared to the controls. When an anode was used as the sole electron acceptor, both the bpfA and bpfG mutations positively impacted mean current density, yielding a 1.8-fold increase for each mutation. [ABSTRACT FROM AUTHOR]

This work has the purpose to elucidate in deeper detail the conjugated physical phenomena at the interface between two immiscible fluids in microfluidic devices. The two typical regimes—dripping and jetting—emerging in flow-focusing devices are considered for the analysis. Dynamic (time-dependent analysis of fixed or Lagrangian-tracked points) and local (lines along the interface, at a fixed time instance) analyses have been conducted from a parallel numerical simulation on a fine numerical grid. The results comprise various pressures and tangential stresses and their balance during the droplet formation process with special attention paid to the moments and locations of the droplet release. It was found that the dripping regime is characterized by the local balance of the pressure drop due to surface tension Δ p σ and the Laplace pressure Δ p Lapl across the interface. Only at the last moments before the droplet pinch-off does the former pressure dominate. In contrast, in the jetting regime, there is a clear domination of the pressure due to tension during the whole process of droplet formation. Shear stresses, presented by the von Mises criterion, are several times (jetting regime) or even an order of magnitude (dripping regime) lower than the surface tension pressure and the Laplace pressure. In both regimes, when the interface curvature κ changes locally its sign, the pressure at the centerline axis shows a clear local maximum. For the jetting regime, the downstream derivative of this centerline pressure is the first parameter that changes along the jet axis—thus indicating the onset of instabilities for this regime—and it is then followed by a wavelike change of the radius of the jet. [ABSTRACT FROM AUTHOR]

As modern electronic devices are increasingly miniaturized and integrated, their performance relies more heavily on effective thermal management. In this regard, two-phase cooling methods which capitalize on thin-film evaporation atop structured porous surfaces are emerging as potential solutions. In such porous structures, the optimum heat dissipation capacity relies on two competing objectives that depend on mass and heat transfer. Optimizing these objectives for effective thermal management is challenging due to the simulation costs and the high dimensionality of the design space which is often a voxelated microstructure representation that must also be manufacturable. We address these challenges by developing a data-driven framework for designing optimal porous microstructures for cooling applications. In our framework, we leverage spectral density functions to encode the design space via a handful of interpretable variables and, in turn, efficiently search it. We develop physics-based formulas to simulate the thermofluidic properties and assess the feasibility of candidate designs based on offline image-based analyses. To decrease the reliance on expensive simulations, we generate multi-fidelity data and build emulators to find Pareto-optimal designs. We apply our approach to a canonical problem on evaporator wick design and obtain fin-like topologies in the optimal microstructures which are also characteristics often observed in industrial applications. [ABSTRACT FROM AUTHOR]

Biosecurity encompasses the health and safety of humans, animals, plants, and the environment. In this article, "biosecurity" is defined as encompassing the comprehensive aspects of human, animal, plant, and environmental safety. Reliable biosecurity testing technology is the key point for effectively assessing biosecurity risks and ensuring biosecurity. Therefore, it is crucial to develop excellent detection technologies to detect risk factors that can affect biosecurity. An electrochemical microfluidic biosensing platform integrates fluid control, target recognition, signal transduction, and output and incorporates the advantages of electrochemical analysis technology and microfluidic technology. Thus, an electrochemical microfluidic biosensing platform, characterized by exceptional analytical sensitivity, portability, rapid analysis speed, low reagent consumption, and low risk of contamination, shows considerable promise for biosecurity detection compared to traditional, more complex, and time-consuming detection technologies. This review provides a concise introduction to electrochemical microfluidic biosensors and biosecurity. It highlights recent research advances in utilizing electrochemical microfluidic biosensing platforms to assess biosecurity risk factors. It includes the use of electrochemical microfluidic biosensors for the detection of risk factors directly endangering biosecurity (direct application: namely, risk factors directly endangering the health of human, animals, and plants) and for the detection of risk factors indirectly endangering biosecurity (indirect application: namely, risk factors endangering the safety of food and the environment). Finally, we outline the current challenges and future perspectives of electrochemical microfluidic biosensing platforms. [ABSTRACT FROM AUTHOR]

Factor XII (FXII) is the zymogen of the plasma protease FXIIa that activates the intrinsic coagulation pathway and the kallikrein kinin-system. The role of FXII in inflammation has been obscure. Here, we report a single-domain antibody (nanobody, Nb) fused to the Fc region of a human immunoglobulin (Nb-Fc) that recognizes FXII in a conformation-dependent manner and interferes with FXIIa formation. Nb-Fc treatment inhibited arterial thrombosis in male mice without affecting hemostasis. In a mouse model of extracorporeal membrane oxygenation (ECMO), FXII inhibition or knockout reduced thrombus deposition on oxygenator membranes and systemic microvascular thrombi. ECMO increased circulating levels of D-dimer, alkaline phosphatase, creatinine and TNF-α and triggered microvascular neutrophil adherence, platelet aggregation and their interaction, which were substantially attenuated by FXII blockade. Both Nb-Fc treatment and FXII knockout markedly ameliorated immune complex-induced local vasculitis and anti-neutrophil cytoplasmic antibody-induced systemic vasculitis, consistent with selectively suppressed neutrophil migration. In human blood microfluidic analysis, Nb-Fc treatment prevented collagen-induced fibrin deposition and neutrophil adhesion/activation. Thus, FXII is an important mediator of inflammatory responses in vasculitis and ECMO, and Nb-Fc provides a promising approach to alleviate thrombo-inflammatory disorders. Thrombosis and inflammation coexist in many diseases, however, there is lack of treatments targeting both pathologies. This study reports a novel antibody against blood factor XII, which bears a promise to treat broad thrombo-inflammatory disorder. [ABSTRACT FROM AUTHOR]

Electrochemical sensors provide an affordable and reliable approach towards the detection and monitoring of important biological species ranging from simple ions to complex biomolecules. The ability to miniaturize electrochemical sensors, coupled with their affordability and simple equipment requirements for signal readout, permits the use of these sensors at the point-of-care where analysis using non-invasively obtainable biofluids is receiving growing interest by the research community. This paper describes the design, fabrication, and integration of a 3D printed Mg2+ potentiometric sensor into a 3D printed microfluidic device for the quantification of Mg2+ in low-sample volume biological fluids. The sensor employs a functionalized 3D printable photocurable methacrylate-based ion-selective membrane affixed to a carbon-mesh/epoxy solid-contact transducer for the selective determination of Mg2+ in sweat, saliva and urine. The 3D printed Mg2+ ion-selective electrode (3Dp-Mg2+-ISE) provided a Nernstian response of 27.5 mV per decade with a linear range of 10 mM to 39 μM, covering the normal physiological and clinically relevant levels of Mg2+ in biofluids. 3Dp-Mg2+-ISEs selectively measure Mg2+ over other biologically present cations – sodium, potassium, calcium, ammonium – as well as provide high stability in the analytical signal with a drift of just 13 μV h−1 over 10 hours. Comparison with poly(vinylchloride)-based Mg2+-ISEs showed distinct advantages to the use of 3Dp-Mg2+-ISEs, with respect to stability, resilience towards biofouling and importantly providing a streamlined and rapid approach towards mass production of selective and reliable sensors. The miniaturization capabilities of 3D printing coupled with the benefits of microfluidic analysis (i.e., low sample volumes, minimal reagent consumption, automation of multiple assays, etc.), provides exciting opportunities for the realization of the next-generation of point-of-care diagnostic devices. [ABSTRACT FROM AUTHOR]

Laboratory sciences and disease diagnostics need accelerated medical tests, ease of testing for patients, and lower diagnostic costs. In this respect, one of the most innovative methods is paper-based microfluidic analysis tools, called micro-PAD. Since the chemical-based micro-PADs need sufficient blood to show an accurate blood test, the shape of the micro-PADs, the blood quantity, and the droplet flow to reach the end of the microchannels are important. Therefore, this research showed the liquid droplet motion on a heterogeneous (hydrophobic and hydrophilic) surface using the lattice Boltzmann method, and two star-shaped micro-PADs were modeled to investigate the effect of the droplet size and the droplet deflection from the center of the micro-PAD on liquid flow in microchannels. Based on the obtained results, fluid behavior was similar in two patterns (ten-pointed pattern and four-pointed pattern). This study compared the effects of parallel, convergent, and divergent channels in a four-pointed pattern on fluid velocity. The off-center error of the droplet was considered at the initial moment. The obtained results confirmed the direct relationship between the increased deviation of the center and the lack of uniformity in the fluid penetration. The present simulation results were validated against the laboratory data. [ABSTRACT FROM AUTHOR]

Average-sized microfluidic artificial lungs consisting of rows and columns of fiber bundles with different column to row aspect ratios (AR) are numerically analyzed for flow characteristics, maximum gas transfer performance, minimum pressure drop, and proper wall shear stress (WSS) values in terms of biocompatibility. The flow is fully laminar and assumed to be incompressible and Newtonian. The transport analysis is performed using a combined convection-diffusion model, and the numerical simulations are carried out with the finite element method. The inlet volumetric flow is modeled as a sinusoidal wave function to simulate the cardiac cycle and its effect on the device performance. The model is first validated with experimental studies in steady-state condition and compared with existing correlations for transient conditions. Then, the validated model is used for a parametric study in both steady and pulsatile flow conditions. The results show that increasing the aspect ratio in fiber configuration leads to converging gas transfer, higher pressure drop, and higher WSS. While determining the optimum configuration, the acceptable shear stress levels play a decisive role to ensure biocompatibility. Also, it is observed that the steady analysis underestimates the gas transfer for higher aspect ratios. [ABSTRACT FROM AUTHOR]

Simple Summary: The development and use of migrastatic drugs could help improve the outcomes for patients with high-grade non-muscle-invasive bladder cancer and muscle-invasive bladder cancer by inhibiting bladder cancer cell invasion and thereby decreasing the likelihood of disease progression and metastasis. A key barrier to this is the lack of suitable models to assess bladder cancer cell invasiveness. Here, we describe a bladder cancer-on-a-chip model. We demonstrate that our model supports the retention of the bladder cancer cell phenotype and can be used to reproducibly assess and quantify the invasiveness of live bladder cancer cells. Treatment with ATN-161, a well-known migrastatic drug, caused a dose-dependent decrease in bladder cancer cell invasiveness thereby validating the ability of our model to test migrastatic drugs. To our knowledge, a bladder cancer-on-a-chip model that can specifically assess bladder cancer invasiveness has not previously been described. We have developed a bladder cancer-on-a-chip model which supports the 3D growth of cells and can be used to assess and quantify bladder cancer cell invasiveness in a physiologically appropriate environment. Three bladder cancer cell lines (T24, J82, and RT4) were resuspended in 50% Matrigel® and grown within a multi-channel organ-on-a-chip system. The ability of live cells to invade across into an adjacent 50% Matrigel®-only channel was assessed over a 2-day period. Cell lines isolated from patients with high-grade bladder cancer (T24 and J82) invaded across into the Matrigel®-only channel at a much higher frequency compared to cells isolated from a patient with low-grade cancer (RT4) (p < 0.001). The T24 and J82 cells also invaded further distances into the Matrigel®-only channel compared to the RT4 cells (p < 0.001). The cell phenotype within the model was maintained as assessed by cell morphology and immunohistochemical analysis of E-cadherin. Treatment with ATN-161, an α5β1 integrin inhibitor and well-known migrastatic drug, caused a dose-dependent decrease in the invasiveness of the J82 cells (p < 0.01). The combined data demonstrate that our bladder cancer-on-a-chip model supports the retention of the bladder cancer cell phenotype and can be used to reproducibly assess and quantify the invasiveness of live bladder cancer cells. [ABSTRACT FROM AUTHOR]

Passive fluid control has mostly been used for valves, pumps, and mixers in microfluidic systems. The basic principle is to generate localized losses in special channel structures, such as branches, grooves, or spirals. The flow field in two-dimensional space can be easily calculated using the typical Stokes formula, but it is challenging in three-dimensional space. Moreover, the flow field with periodic variable cross-sections channeled of polyhedral units has been neglected in this research field due to previous limitations in manufacturing technology. With the continuous progress of 3D printing technology, the field of microfluidic devices ushered in a new era of manufacturing three-dimensional irregular channels. In this study, we present finite analysis results for a periodic nodular-like channel. The experiments involve variations in the Reynold number (Re), periodic frequency, and comparative analyses with conventional structures. The findings indicate that this variable 3D cross-section structure can readily achieve performance comparable to other passive fluid control methods in valve applications. A 3D model of the periodic tetrahedron channel was fabricated using 3D printing to validate these conclusions. This research has the potential to significantly enhance the performance of passive fluid control units that have long been constrained by manufacturing dimensions. [ABSTRACT FROM AUTHOR]

This paper aims to establish a panoramic foundation for investigating the impact of sunlight on a recently formulated water-based tri-nano hybrid Sutterby liquid (TNHF) under provided magnetic field, directed through photovoltaic solar panels by exploiting the knacks of machine intelligent computing paradigm. The comparative performance of hybrid and tri-nano fluidic system with water as a base fluid is exhaustively analyzed and discussed. This research is unique, as it has a distinguished figurative comparison analysis between two different types of nanofluidic materials, which helps to choose the best one for use in agrivoltaics systems, and replace the materials already in use to enhance the efficiency and performance coefficients. Furthermore, the description of the composition scheme makes this research more feasible and applicable. A numerical dataset of nonlinear mathematical model is generated by employing finite difference scheme in the recently introduced Python bvp-solver algorithm, then it is embedded into artificial intelligence (AI)-based Levenberg Marquardt neural network algorithm (LMNNA). A significant outcome of the research indicates that the integration TNHF results in a notably faster enhancement of heat transfer rate and temperature framework as compared to traditional hybrid fluid. It is observed that introducing three distinct nanomaterials of specific thermophysical characteristics enhances the thermal exchanging profile and faces an obvious flow rate dissipation in solar plate channels. The standard numerical and AI-generated results are documented to portray the stability, accuracy and efficacy of scheme in terms of iterative learning curves on MSE, error analysis, histograms and regression statistics. Additional perquisites of the methodology include cost effectiveness, time-saving ability, robustness, stability and its extendibility. [ABSTRACT FROM AUTHOR]

Cell-cell interactions in the central nervous system play important roles in neurologic diseases. However, little is known about the specific molecular pathways involved, and methods for their systematic identification are limited. Here, we developed a forward genetic screening platform that combines CRISPR-Cas9 perturbations, cell coculture in picoliter droplets, and microfluidic-based fluorescence-activated droplet sorting to identify mechanisms of cell-cell communication. We used SPEAC-seq (systematic perturbation of encapsulated associated cells followed by sequencing), in combination with in vivo genetic perturbations, to identify microglia-produced amphiregulin as a suppressor of disease-promoting astrocyte responses in multiple sclerosis preclinical models and clinical samples. Thus, SPEAC-seq enables the high-throughput systematic identification of cell-cell communication mechanisms.

Recent years have seen continued expansion of the functionality of lab on a chip (LOC) devices. Indeed LOCs now provide scientists and developers with useful and versatile platforms across a myriad of chemical and biological applications. The field still fails, however, to integrate an often important element of bench-top analytics: real-time molecular measurements that can be used to guide a chemical response. Here we describe the analytical techniques that could provide LOCs with such real-time molecular monitoring capabilities. It appears to us that, among the approaches that are general (i.e., that are independent of the reactive or optical properties of their targets), sensing strategies relying on binding-induced conformational change of bioreceptors are most likely to succeed in such applications.

Numerous immunoassays have been successfully integrated on disc-based centrifugal platforms (CDs) over the last 20 years. These CD devices can be used as portable point-of-care (POC) platforms with sample-to-answer capabilities where bodily fluids such as whole blood can be used as samples directly without pre-processing. In order to use whole blood as a sample on CDs, centrifugation is used to separate red blood cells from plasma on CDs. There are several techniques for using specific fluidic patterns in the centrifugal fluidic network, such as reciprocation, that enhances the sensitivity of the immunoassays, including those using microarray antigen membranes. Present work demonstrates, for the first time, simultaneous integration of blood plasma separation (BPS) and reciprocation on the CD platform. The integrated design allows plasma that is separated from the red blood cells in a sedimentation chamber to flow into the reciprocation chamber via a narrow connecting channel of 0.5 mm × 0.5 mm cross-section. Due to the small cross-section of the connecting channel, there is no inflow of the red blood cell into the reciprocation chamber during subsequent fluidic operations of the CD. While no inflow of the red blood cells into the reciprocation chamber was observed, the conditions of 20 g jerk acceleration were also simulated in ANSYS finite element analysis software, and it was found that the CD design that was used is capable of retaining red blood cells in the sedimentation chamber. Experimentally, the isolation of red blood cells in the sedimentation chamber was confirmed using the ImageJ image processor to detect the visible color-based separation of the plasma from the blood. A fluorescent analyte testing on the bio-sensing array of the presented novel integrated design and on the standard reciprocation design CD was conducted for 7 min of reciprocation in each case. The test analyte was Europium Streptavidin Polystyrene analyte (10-3 mg/mL) and the microarray consisted of Biotin bovine serum albumin (BSA) dots. The fluorescent signals for the standard and integrated designs were nearly identical (within the margin of error) for the first several minutes of reciprocation, but the fluorescent signal for the integrated design was significantly higher when the reciprocation time was increased to 7 min.

The utilizations of microfluidic chips for single RBC (red blood cell) studies have attracted great interests in recent years to filter, trap, analyze, and release single erythrocytes for various applications. Researchers in this field have highlighted the vast potential in developing micro devices for industrial and academia usages, including lab-on-a-chip and organ-on-a-chip systems. This article critically reviews the current state-of-the-art and recent advances of microfluidics for single RBC analyses, including integrated sensors and microfluidic platforms for microscopic/tomographic/spectroscopic single RBC analyses, trapping arrays (including bifurcating channels), dielectrophoretic and agglutination/aggregation studies, as well as clinical implications covering cancer, sepsis, prenatal, and Sickle Cell diseases. Microfluidics based RBC microarrays, sorting/counting and trapping techniques (including acoustic, dielectrophoretic, hydrodynamic, magnetic, and optical techniques) are also reviewed. Lastly, organs on chips, multi-organ chips, and drug discovery involving single RBC are described. The limitations and drawbacks of each technology are addressed and future prospects are discussed.

Benefiting from the complex system composed of various constituents, medicament portions, species, and places of origin, traditional Chinese medicine (TCM) possesses numerous customizable and adaptable efficacies in clinical practice guided by its theories. However, these unique features are also present challenges in areas such as quality control, screening active ingredients, studying cell and organ pharmacology, and characterizing the compatibility between different Chinese medicines. Drawing inspiration from the holistic concept, an integrated strategy and pattern more aligned with TCM research emerges, necessitating the integration of novel technology into TCM modernization. The microfluidic chip serves as a powerful platform for integrating technologies in chemistry, biology, and biophysics. Microfluidics has given rise to innovative patterns like lab-on-a-chip and organoids-on-a-chip, effectively challenging the conventional research paradigms of TCM. This review provides a systematic summary of the nature and advanced utilization of microfluidic chips in TCM, focusing on quality control, active ingredient screening/separation, pharmaceutical analysis, and pharmacological/toxicological assays. Drawing on these remarkable references, the challenges, opportunities, and future trends of microfluidic chips in TCM are also comprehensively discussed, providing valuable insights into the development of TCM. [ABSTRACT FROM AUTHOR]

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

The exploration of our solar system to characterize the molecular organic inventory will enable the identification of potentially habitable regions and initiate the search for biosignatures of extraterrestrial life. However, it is challenging to perform the required high-resolution, high-sensitivity chemical analyses in space and in planetary environments. To address this challenge, we have developed a microfluidic organic analyzer (MOA) instrument that consists of a multilayer programmable microfluidic analyzer (PMA) for fluidic processing at the microliter scale coupled with a microfabricated glass capillary electrophoresis (CE) wafer for separation and analysis of the sample components. Organic analytes are labeled with a functional group-specific (e.g. amine, organic acid, aldehyde) fluorescent dye, separated according to charge and hydrodynamic size by capillary electrophoresis (CE), and detected with picomolar limit of detection (LOD) using laser-induced fluorescence (LIF). Our goal is a sensitive automated instrument and autonomous process that enables sample-in to data-out performance in a flight capable format. We present here the design, fabrication, and operation of a technology development unit (TDU) that meets these design goals with a core mass of 3 kg and a volume of <5 L. MOA has a demonstrated resolution of 2 × 105 theoretical plates for relevant amino acids using a 15 cm long CE channel and 467 V cm-1. The LOD of LIF surpasses 100 pM (0.01 ppb), enabling biosignature detection in harsh environments on Earth. MOA is ideally suited for probing biosignatures in potentially habitable destinations on icy moons such as Europa and Enceladus, and on Mars. [ABSTRACT FROM AUTHOR]

The global impact of cancer on human health has raised significant concern. In this context, the tumor microenvironment (TME) plays a pivotal role in the tumorigenesis and malignant progression. In order to enhance the accuracy and efficacy of therapeutic outcomes, there is an imminent requirement for in vitro models that can accurately replicate the intricate characteristics and constituents of TME. Microfluidic devices exhibit notable advantages in investigating the progression and treatment of tumors and have the potential to become a novel methodology for evaluating immune cell activities in TME and assist clinicians in assessing the prognosis of patients. In addition, it shows great advantages compared to traditional cell experiments. Therefore, the review first outlines the applications and advantages of microfluidic chips in facilitating tumor cell culture, constructing TME and investigating immune cell activities. Second, the roles of microfluidic devices in the analysis of circulating tumor cells, tumor prognosis, and drug screening have also been mentioned. Moreover, a forward-looking perspective is discussed, anticipating the widespread clinical adoption of microfluidic devices in the future. [ABSTRACT FROM AUTHOR]

Delayed cerebral ischemia (DCI) occurs in up to one third of patients suffering from aneurysmal subarachnoid hemorrhage (aSAH). Untreated, it leads to secondary cerebral infarctions and is frequently associated with death or severe disability. After aneurysm rupture, erythrocytes in the subarachnoid space lyse and liberate free hemoglobin (Hb), a key driver for the development of DCI. Hemoglobin in the cerebrospinal fluid (CSF-Hb) can be analyzed through a two-step procedure of centrifugation to exclude intact erythrocytes and subsequent spectrophotometric quantification. This analysis can only be done in specialized laboratories but not at the bedside in the intensive care unit. This limits the number of tests done, increases the variability of the results and restricts accuracy. Bedside measurements of CSF-Hb as a biomarker with a point of care diagnostic test system would allow for a continuous monitoring for the risk of DCI in the individual patient. In this study, a microfluidic chip was explored that allows to continuously separate blood particles from CSF or plasma based on acoustophoresis. An in vitro test bench was developed to test in-line measurements with the developed microfluidic chip and a spectrometer. The proof of principle for a continuous particle separation device has been established with diluted blood and CSF samples from animals and aSAH patients, respectively. Processing 1 mL of blood in our microfluidic device was achieved within around 70 min demonstrating only minor deviations from the gold standard centrifugation (7% average error of patient samples), while saving several hours of processing time and additionally the reduction of deviations in the results due to manual labor. [ABSTRACT FROM AUTHOR]

In the field of tissue engineering, local hypoxia in large-cell structures (larger than 1 mm3) poses a significant challenge. Oxygen-releasing biomaterials supply an innovative solution through oxygen ⁠ delivery in a sustained and controlled manner. Compared to traditional methods such as emulsion, sonication, and agitation, microfluidic technology offers distinct benefits for oxygen-releasing material production, including controllability, flexibility, and applicability. It holds enormous potential in the production of smart oxygen-releasing materials. This review comprehensively covers the fabrication and application of microfluidic-enabled oxygen-releasing biomaterials. To begin with, the physical mechanism of various microfluidic technologies and their differences in oxygen carrier preparation are explained. Then, the distinctions among diverse oxygen-releasing components in regards for oxygen-releasing mechanism, oxygen-carrying capacity, and duration of oxygen release are presented. Finally, the present obstacles and anticipated development trends are examined together with the application outcomes of oxygen-releasing biomaterials based on microfluidic technology in the biomedical area. Oxygen is essential for sustaining life, and hypoxia (a condition of low oxygen) is a significant challenge in various diseases. Microfluidic-based oxygen-releasing biomaterials offer precise control and outstanding performance, providing unique advantages over traditional approaches for tissue engineering. However, comprehensive reviews on this topic are currently lacking. In this review, we provide a comprehensive analysis of various microfluidic technologies and their applications for developing oxygen-releasing biomaterials. We compare the characteristics of organic and inorganic oxygen-releasing biomaterials and highlight the latest advancements in microfluidic-enabled oxygen-releasing biomaterials for tissue engineering, wound healing, and drug delivery. This review may hold the potential to make a significant contribution to the field, with a profound impact on the scientific community. [Display omitted] [ABSTRACT FROM AUTHOR]

The handling and analysis of gaseous tritium is of interest for hydrogen isotope separation experiments. In this work, we present an easy-to-handle setup for catalytic oxidation to HTO, recovering all of the initially dosed gaseous tritium as determined by LSC, using CuO as a catalyst at a reaction temperature of 900 °C. Aiming to reduce cocktail waste, the LSC determination was downscaled to a microfluidic setup. The performance was evaluated based on the counting efficiency, which was shown to decrease significantly, as the sample volume was reduced to µl amounts, while no changes were observed over a wide range of sample-to-cocktail ratios. [ABSTRACT FROM AUTHOR]

The GeneSoC® that launched recently enables the quantitative detection of target genes (in approximately 15 min) using microfluidic thermal cycling technology. Here, we compared the diagnostic performance of the "GeneSoC® SARS-CoV-2 N2 Detection Kit" (Kyorin assay) and conventional severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection assays to verify the clinical usefulness of the Kyorin assay. Two hundred samples (100 nasopharyngeal and 100 saliva specimens) were collected from patients with suspected SARS-CoV-2 infection between May 2020 and August 2021. Conventional SARS-CoV-2 detection assays were performed using the Japanese National Institute of Infectious Diseases (NIID) assay, Ampdirect™ 2019-nCoV Detection Kit (SHIMADZU assay), and Lumipulse Presto SARS-CoV-2 Ag (FUJIREBIO assay), according to each manufacturer's instructions. Using the NIID and SHIMADZU assays as references, the positive and negative concordance rates and the kappa coefficient in the Kyorin assay were 96.9–97.9 %, 99.0–100.0 %, and 0.96–0.98, respectively. The positivity rate of the FUJIREBIO assay was slightly lower than that of the reference assay (p<0.05). The Kyorin assay showed a favorable concordance rate with conventional SARS-CoV-2 detection assays, making it a useful, rapid, and high-performance assay for detecting SARS-CoV-2, which may lead to early diagnosis and appropriate treatment. [ABSTRACT FROM AUTHOR]

In drop-based microfluidics, an aqueous sample is partitioned into drops using individual pump sources that drive water and oil into a drop-making device. Parallelization of drop-making devices is necessary to achieve high-throughput screening of multiple experimental conditions, especially in time-sensitive studies. Here, we present the plate-interfacing parallel encapsulation (PIPE) chip, a microfluidic chip designed to generate 50 to 90 μm diameter drops of up to 96 different conditions in parallel by interfacing individual drop makers with a standard 384-well microtiter plate. The PIPE chip is used to generate two types of optically barcoded drop libraries consisting of two-color fluorescent particle combinations: a library of 24 microbead barcodes and a library of 192 quantum dot barcodes. Barcoded combinations in the drop libraries are rapidly measured within a microfluidic device using fluorescence detection and distinct barcoded populations in the fluorescence drop data are identified using DBSCAN data clustering. Signal analysis reveals that particle size defines the source of dominant noise present in the fluorescence intensity distributions of the barcoded drop populations, arising from Poisson loading for microbeads and shot noise for quantum dots. A barcoded population from a drop library is isolated using fluorescence-activated drop sorting, enabling downstream analysis of drop contents. The PIPE chip can improve multiplexed high-throughput assays by enabling simultaneous encapsulation of barcoded samples stored in a microtiter plate and reducing sample preparation time.

Superhydrophobic surface-based optofluidics have been introduced to biosensors and unconventional optics with unique advantages, such as low light loss and power consumption. However, most of these platforms were made with planar-like microstructures and nanostructures, which may cause bonding issues and result in significant waveguide loss. Here, we introduce a fully enclosed superhydrophobic-based optofluidics system, enabled by a one-step microstereolithography procedure. Various microstructured cladding designs with a feature size down to 100 μm were studied and a T-type overhang design exhibits the lowest optical loss, regardless of the excitation wavelength. Surprisingly, the optical loss of superhydrophobic-based optofluidics is not solely decided by the solid area fraction at the solid/water/air interface, but also the cross-section shape and the effective cladding layer composition. We show that this fully enclosed optofluidic system can be used for CRISPR-labeled quantum dot quantification, intended for in vitro and in vivo CRISPR therapeutics.

Protein networks can be assembled in vitro for basic biochemistry research, drug screening, and the creation of artificial cells. Two standard methodologies are used: manual pipetting and pipetting robots. Manual pipetting has limited throughput in the number of input reagents and the combination of reagents in a single sample. While pipetting robots are evident in improving pipetting efficiency and saving hands-on time, their liquid handling volume usually ranges from a few to hundreds of microliters. Microfluidic methods have been developed to minimize the reagent consumption and speed up screening but are challenging in multifactorial protein studies due to their reliance on complex structures and labeling dyes. Here, we engineered a new impact-printing-based methodology to generate printed microdroplet arrays containing water-in-oil droplets. The printed droplet volume was linearly proportional (R2 = 0.9999) to the single droplet number, and each single droplet volume was around 59.2 nL (coefficient of variation = 93.8%). Our new methodology enables the study of protein networks in both membrane-unbound and -bound states, without and with anchor lipids DGS-NTA(Ni), respectively. The methodology is demonstrated using a subnetwork of mitogen-activated protein kinase (MAPK). It takes less than 10 min to prepare 100 different droplet-based reactions, using

The versatility, rapid development, and ease of production scalability of mRNA therapeutics have placed them at the forefront of biopharmaceutical research. However, despite their vast potential to treat diseases, their novelty comes with unsolved analytical challenges. A key challenge in ensuring sample purity has been monitoring residual, immunostimulatory dsRNA impurities generated during the in vitro transcription of mRNA. Here, we present a method that combines an enzyme, S1 nuclease, to identify and isolate dsRNA from an mRNA sample with a microfluidic electrophoresis analytical platform to characterize the impurity. After the method was developed and optimized, it was tested with clinically relevant, pseudouridine-modified 700 and 1800 bp dsRNA and 818–4451 nt mRNA samples. While the treatment impacted the magnitude of the fluorescent signal used to analyze the samples due to the interference of the buffer with the labeling of the sample, this signal loss was mitigated by 8.8× via treatment optimization. In addition, despite the mRNA concentration being up to 400× greater than that of the dsRNA, under every condition, there was a complete disappearance of the main mRNA peak. While the mRNA peak was digested, the dsRNA fragments remained physically unaffected by the treatment, with no change to their migration time. Using these samples, we detected 0.25% dsRNA impurities in mRNA samples using 15 μL with an analytical runtime of 1 min per sample after digestion and were able to predict their size within 8% of the expected length. The short runtime, sample consumption, and high throughput compatibility make it suitable to support the purity assessment of mRNA during purification and downstream. [ABSTRACT FROM AUTHOR]

The manipulation of magnetic microparticles has always been pivotal in the development of microfluidic devices, as it encompasses a broad range of applications, such as drug delivery, bioanalysis, on-chip diagnostics, and more recently organ-on-chip development. However, predicting the behavior and trajectory of these particles remains a recurring and partly unresolved question. Magnetic particle-laden flows can display intricate collective behaviors, such as packed plugs, column-shaped aggregates, or fluidization, which are difficult to predict. In this study, we introduce a finite-element model to simulate highly dense flows of magnetic microparticles. Our method relies on an interpenetrating continuum approach, where both the liquid and particle phases are described by the Navier–Stokes equations, in which the magnetic force, interphase friction, and interparticle forces were included. We demonstrate its applicability across the entire range of particle packing densities and compare the results with experimental data from real microfluidic application cases. The model successfully replicates complex behaviors, such as particle aggregation, plug formation and fluidization. This approach has potential to accelerate microfluidic device development by reducing the need for costly and time-consuming experimental optimization. [ABSTRACT FROM AUTHOR]

The riparian zone, found alongside rivers and streams, is a unique habitat characterized by its vulnerability to sudden floods following intense rainfall. To cope with these challenging conditions, a specific group of plants with linear and lanceolate lamina have adapted to thrive in these environments. Despite their unique ability to withstand the forceful water flow, the specific adaptive characteristics of the petioles, which support the lamina remain unknown. Our morphological, anatomical, and mechanical analyses on the petioles of Osmunda lancea (Osmundaceae) along the river and an inland sister species of O. japonica revealed that the petioles of O. lancea had a larger cell volume in subepidermal cortex and were more flexible than those of O. japonica. [ABSTRACT FROM AUTHOR]

• Linear cooling of continuous microfluidic was achieved based on a single TEC. • Multi-structure TEC was proposed to generate a linear temperature gradient. • Numerical model was established and verified by experiment. • Linearity, cooling rate and temperature of linear cooling were analyzed. • Cooling rate can be controlled by changing the current and microfluidic velocity. Linear cooling of continuous microfluidic is crucial to the microfluidic analysis of cell cryopreservation and homogenous ice nucleation. This study presents a method to establish a linear temperature gradient for linear cooling of continuous microfluidic based on a single thermoelectric cooler. A novel multi-structure thermoelectric cooler was proposed to generate a stable linear temperature gradient. Furthermore, a numerical model was established to explore the performance of linear cooling of continuous microfluidic. Meanwhile, experiments were carried out to validate the model. Based on the proposed multi-structure thermoelectric cooler, linear cooling of continuous microfluidic can be successfully achieved, even if the current of the thermoelectric cooler or microfluidic velocity changes. For the case of current equal to 3 A and microfluidic velocity equal to 50 mm/s, microfluidic can be linearly cooled from 297 K to 251 K with a cooling rate of 64.5 K/s and linearity of 0.9912. It was also found that the temperature gradient decreases slightly with increasing microfluidic velocity while the cooling rate increases and is approximately proportional to the microfluidic velocity. With the increase of microfluidic velocity , the linearity increases at first and then decreases. Maximum goodness of fit equal to 0.9942 appears at the microfluidic velocity of 20 mm/s. [ABSTRACT FROM AUTHOR]

Background: The functional heterogeneity of culture-expanded mesenchymal stem cells (MSCs) has hindered the clinical application of MSCs. Previous studies have shown that MSC subpopulations with superior chondrogenic capacity can be isolated using a spiral microfluidic device based on the principle of inertial cell focusing. Hypothesis: The delivery of microfluidic-enriched chondrogenic MSCs that are consistent in size and function will overcome the challenge of the functional heterogeneity of expanded MSCs and will significantly improve MSC-based cartilage repair. Study Design: Controlled laboratory study. Methods: A next-generation, fully automated multidimensional double spiral microfluidic device was designed to provide more refined and efficient isolation of MSC subpopulations based on size. Analysis of in vitro chondrogenic potential and RNA sequencing was performed on size-sorted MSC subpopulations. In vivo cartilage repair efficacy was demonstrated in an osteochondral injury model in 12-week-old rats. Defects were implanted with MSC subpopulations (n = 6 per group) and compared with those implanted with unsegregated MSCs (n = 6). Osteochondral repair was assessed at 6 and 12 weeks after surgery by histological, micro–computed tomography, and mechanical analysis. Results: A chondrogenic MSC subpopulation was efficiently isolated using the multidimensional double spiral device. RNA sequencing revealed distinct transcriptomic profiles and identified differential gene expression between subpopulations. The delivery of a chondrogenic MSC subpopulation resulted in improved cartilage repair, as indicated by histological scoring, the compression modulus, and micro–computed tomography of the subchondral bone. Conclusion: We have established a rapid, label-free, and reliable microfluidic protocol for more efficient size-based enrichment of a chondrogenic MSC subpopulation. Our proof-of-concept in vivo study demonstrates the enhanced cartilage repair efficacy of these enriched chondrogenic MSCs. Clinical Relevance: The delivery of microfluidic-enriched chondrogenic MSCs that are consistent in size and function can overcome the challenge of the functional heterogeneity of expanded MSCs, resulting in significant improvement in MSC-based cartilage repair. The availability of such rapid, label-free enriched chondrogenic MSCs can enable better cell therapy products for cartilage repair with improved treatment outcomes. [ABSTRACT FROM AUTHOR]

From their initial application to the fields of chemistry and biology, microfluidic chips used as micro total analysis systems have developed into new technologies to satisfy the requirements of various societal industries. Microchannels are essential components of microfluidic chips; they play a vital role in connecting the inlet and outlet as well as determining the flow distribution and reagent mixing. Microfluidic research has always been devoted to minimizing energy dissipation, fluid resistance, and pressure drop to realize energy-efficient microfluidic chips. This study proposes a new theory for manipulating the flow in microchannels based on hydrodynamic metamaterials according to the spatial transformation theory. In particular, hydrodynamic metamaterials are specifically designed to construct flow shifters, flow splitters, and flow combiners, and theoretical and numerical simulations are performed to assess their hydrodynamic performance. The systematic design of hydrodynamic metamaterial devices proposed in this work establishes a theoretical framework to achieve a steady flow state without inducing unstable flow disturbances in complex-shape microchannels. [ABSTRACT FROM AUTHOR]

Single-cell encapsulation in droplet microfluidics is commonly hindered by the tradeoff between cell suspension density and on-chip focusing performance. In this study, we introduce a novel droplet microfluidic chip to overcome this challenge. The chip comprises a double spiral focusing unit, a flow resistance-based sample enrichment module with fine-tunable outlets, and a crossflow droplet generation unit. Utilizing a low-density cell/bead suspension (2 × 106 objects/mL), cells/beads are focused into a near-equidistant linear arrangement within the double spiral microchannel. The excess water phase is diverted while cells/beads remain focused and sequentially encapsulated in individual droplets. Focusing performance was assessed through numerical simulations and experiments at three flow rates (40, 60, 80 μL/min), demonstrating successful focusing at 40 and 80 μL/min for beads and cells, respectively. In addition, both simulation and experimental results revealed that the flow resistance at the sample enrichment module is adjustable by punching different outlets, allowing over 50% of the aqueous phase to be removed. YOLOv8n-based droplet detection algorithms realized the counting of cells/beads in droplets, statistically demonstrating single-cell and bead encapsulation rates of 72.2% and 79.2%, respectively. All the results indicate that this on-chip sample enrichment approach can be further developed and employed as a critical component in single-cell encapsulation in water-in-oil droplets. [ABSTRACT FROM AUTHOR]

Mixing of the algal culture medium in a photobioreactor (PBR) is a crucial factor to enhance microalgae cultivation and production, since it ensures microalgae cells can homogeneously access nutrients and light radiation. Hydrodynamic conditions in an industrially applicable PBR cannot be tested in laboratory cultivation systems since the scale-up methodology is not specified; hence requiring complex tests at a demonstrative scale or the application of mathematical models. In this study, a three-dimensional numerical model of the hydrodynamic conditions in a hybrid horizontal tubular PBR was developed to investigate the mixing of the culture medium under various operating conditions, in order to optimize biomass production in this type of system. The PBR consists of two open tanks connected through sixteen polyethylene transparent tubes, at a demonstrative scale (total volume 11.7 m3). Microalgae cells movement inside the tubes was simulated. Cell trajectory prediction allows to simulate the intensity of mixing of the culture medium. Subsequent coupling with the biokinetic BIO_ALGAE model allows to monitor the influence of hydrodynamic conditions on the distribution of light in the culture medium and the yield of microalgae. The simulations were experimentally validated. The more intense mixing of the culture medium caused by increasing flow rate (Re = 23,700–46,200) allows more frequent exposure of the microalgae cells to the light source, which according to the results obtained can increase the microalgal yield by 14% in the summer cultivation period and by 151% in the winter period. [Display omitted] [ABSTRACT FROM AUTHOR]

Micromixers play a crucial role as essential components in microfluidic analysis systems. This paper introduces a novel micromixer designed by harnessing electrokinetic vortices arising on the surface of a Janus droplet within a microchannel. The Janus droplet is characterized by different polarities of charges on its two sides (upstream part and downstream part). In the presence of a direct current electric field, the droplet's surface generates electroosmotic flows in opposite directions, resulting in the formation of vortices and facilitating solution mixing. Results from numerical simulations suggest that a better mixing performance of the micromixer is associated with both a higher absolute value of the zeta potential ratio between the downstream and upstream surfaces of the Janus droplet and a larger downstream surface area. Additionally, this study reveals that microchannel dimensions significantly influence the performance of the micromixer. Smaller microchannel widths and heights correspond to a larger mixing index for the micromixer. The micromixer presented in this study features a simple structure, easy fabrication, and holds promising application potential. [ABSTRACT FROM AUTHOR]

Two spectrophotometric methods have been developed for the determination of methyldopa in the pure form and pharmaceutical formulations, both two methods based on the oxidation of the drug with an excess of N-Bromosuccinimide (NBS) and then reduction with 3,3-Diaminobenzidine (DAB), Absorbance of the resulting Magenta colored product is measured at 513 nm, the linearity ranged between (0.5 to 10) mg L-1 for the first spectroscopy method, and (0.5 to 15) mg L-1 for the second microfluid method. The detection limits (LOD) are 0.171, and 0.180 µg mL-1 for methyldopa in two methods spectroscopies, and microfluidic respectively. The limits of quantities (LOQ) are 0.571, and 0.600 µg mL-1 for methyldopa in two methods spectroscopies, and microfluidic respectively. The molar absorptivity (Ɛ) 2.58 ×104, 2.112×103 L moL-1 cm-1 for methyldopa in two methods spectroscopies, and microfluidic respectively. No interference was observed from common excipients in formulations. The results show a simple, accurate, fast, and readily applied method to the determination of methyldopa in pharmaceutical products. The proposed method was applied successfully for the determination of the drug in their pharmaceutical formulations. [ABSTRACT FROM AUTHOR]

This paper investigates the physical layer security (PLS) performance of full-duplex cooperative non-orthogonal multiple access (FD-CNOMA) network in the presence of an external passive eavesdropper. Firstly, we derive approximate analytical expressions for the secrecy outage probabilities (SOPs) of the downlink users and the system SOP (SSOP) of a single-cell FD-CNOMA network, considering the presence/absence of direct link from the base station (BS) to the far user, under imperfect successive interference cancellation conditions. We consider both perfect channel state information and imperfect CSI conditions. To enhance the PLS performance, we propose an artificial noise (AN)-aided framework and derive approximate analytical expressions for the SOPs of the downlink users and the SSOP of the AN-aided FD-CNOMA network. The asymptotic SOP and SSOP expressions are also derived, which are used to determine the secrecy diversity orders. The proposed AN-aided framework significantly reduces the SOPs of the users and the SSOP of the network compared to that without AN. To further enhance the PLS performance, we use the Polak-Ribiere conjugate gradient method to determine the optimal power allocation coefficient (OPAC) for the users at the BS that minimizes the SSOP of the AN-aided FD-CNOMA network. The proposed OPAC significantly reduces the SOPs of the users and the SSOP, compared to random selection/equal setting of the PACs. Finally, we extend the performance evaluations to a multi-cell scenario and demonstrate that the PLS performance of the FD-CNOMA network significantly deteriorates in the presence of co-channel interference. [ABSTRACT FROM AUTHOR]

Microfluidic devices are widely used in many fields of biology, but a key limitation is that cells are typically surrounded by solid walls, making it hard to access those that exhibit a specific phenotype for further study. Here, we provide a general and flexible solution to this problem that exploits the remarkable properties of microfluidic circuits with fluid walls─transparent interfaces between culture media and an immiscible fluorocarbon that are easily pierced with pipets. We provide two proofs of concept in which specific cell subpopulations are isolated and recovered: (i) murine macrophages chemotaxing toward complement component 5a and (ii) bacteria (Pseudomonas aeruginosa) in developing biofilms that migrate toward antibiotics. We build circuits in minutes on standard Petri dishes, add cells, pump in laminar streams so molecular diffusion creates attractant gradients, acquire time-lapse images, and isolate desired subpopulations in real time by building fluid walls around migrating cells with an accuracy of tens of micrometers using 3D printed adaptors that convert conventional microscopes into wall-building machines. Our method allows live cells of interest to be easily extracted from microfluidic devices for downstream analyses.

Current methods for fabricating microparticles offer limited control over size and shape. Here, we demonstrate a droplet microfluidic method to form polyhedral microparticles with controlled concavity. By manipulating Laplace pressure, buoyancy, and particle rheology, we generate microparticles with diverse shapes and curvatures. Additionally, we demonstrate the particles provide increased capture efficiency when used for particle-templated emulsification. Our approach enables microparticles with enhanced chemical and biological functionality.

Droplet-based microfluidic systems have been employed to manipulate discrete fluid volumes with immiscible phases. Creating the fluid droplets at microscale has led to a paradigm shift in mixing, sorting, encapsulation, sensing, and designing high throughput devices for biomedical applications. Droplet microfluidics has opened many opportunities in microparticle synthesis, molecular detection, diagnostics, drug delivery, and cell biology. In the present review, we first introduce standard methods for droplet generation (i.e. passive and active methods) and discuss the latest examples of emulsification and particle synthesis approaches enabled by microfluidic platforms. Then, the applications of droplet-based microfluidics in different biomedical applications are detailed. Finally, a general overview of the latest trends along with the perspectives and future potentials in the field are provided.

Waterborne diseases cause millions of deaths worldwide, especially in developing communities. The monitoring and rapid detection of microbial pathogens in water is critical for public health protection. This study reports the development of a proof-of-concept portable pathogen analysis system (PPAS) that can detect bacteria in water with the potential application in a point-of-sample collection setting. A centrifugal microfluidic platform is adopted to integrate bacterial cell lysis in water samples, nucleic acid extraction, and reagent mixing with a droplet digital loop mediated isothermal amplification assay for bacteria quantification onto a single centrifugal disc (CD). Coupled with a portable "CD Driver" capable of automating the assay steps, the CD functions as a single step bacterial detection "lab" without the need to transfer samples from vial-to-vial as in a traditional laboratory. The prototype system can detect Enterococcus faecalis, a common fecal indicator bacterium, in water samples with a single touch of a start button within 1 h and having total hands-on-time being less than 5 min. An add-on bacterial concentration cup prefilled with absorbent polymer beads was designed to integrate with the pathogen CD to improve the downstream quantification sensitivity. All reagents and amplified products are contained within the single-use disc, reducing the opportunity of cross contamination of other samples by the amplification products. This proof-of-concept PPAS lays the foundation for field testing devices in areas needing more accessible water quality monitoring tools and are at higher risk for being exposed to contaminated waters.

Cell-cell interactions are important to numerous biological systems, including tissue microenvironments, the immune system, and cancer. However, current methods for studying cell combinations and interactions are limited in scalability, allowing just hundreds to thousands of multicell assays per experiment; this limited throughput makes it difficult to characterize interactions at biologically relevant scales. Here, we describe a paradigm in cell interaction profiling that allows accurate grouping of cells and characterization of their interactions for tens to hundreds of thousands of combinations. Our approach leverages high-throughput droplet microfluidics to construct multicellular combinations in a deterministic process that allows inclusion of programmed reagent mixtures and beads. The combination droplets are compatible with common manipulation and measurement techniques, including imaging, barcode-based genomics, and sorting. We demonstrate the approach by using it to enrich for chimeric antigen receptor (CAR)-T cells that activate upon incubation with target cells, a bottleneck in the therapeutic T cell engineering pipeline. The speed and control of our approach should enable valuable cell interaction studies.

Production of high-energy lipids by microalgae may provide a sustainable energy source that can help tackle climate change. However, microalgae engineered to produce more lipids usually grow slowly, leading to reduced overall yields. Unfortunately, culture vessels used to select cells based on growth while maintaining high biomass production, such as well plates, water-in-oil droplet emulsions, and nanowell arrays, do not provide production-relevant environments that cells experience in scaled-up cultures (e.g., bioreactors or outdoor cultivation farms). As a result, strains that are developed in the laboratory may not exhibit the same beneficial phenotypic behavior when transferred to industrial production. Here, we introduce PicoShells, picoliter-scale porous hydrogel compartments, that enable >100,000 individual cells to be compartmentalized, cultured in production-relevant environments, and selected based on growth and bioproduct accumulation traits using standard flow cytometers. PicoShells consist of a hollow inner cavity where cells are encapsulated and a porous outer shell that allows for continuous solution exchange with the external environment. PicoShells allow for cell growth directly in culture environments, such as shaking flasks and bioreactors. We experimentally demonstrate that Chlorella sp., Saccharomyces cerevisiae, and Chinese hamster ovary cells, used for bioproduction, grow to significantly larger colony sizes in PicoShells than in water-in-oil droplet emulsions (P < 0.05). We also demonstrate that PicoShells containing faster dividing and growing Chlorella clonal colonies can be selected using a fluorescence-activated cell sorter and regrown. Using the PicoShell process, we select a Chlorella population that accumulates chlorophyll 8% faster than does an unselected population after a single selection cycle.

Cell-free protein synthesis can enable the combinatorial screening of many different components and concentrations. However, manual pipetting methods are unfit to handle many cell-free reactions. Here, we describe a microfluidic method that can generate hundreds of unique submicroliter scale reactions. The method is coupled with a high yield cell-free system that can be applied for broad protein screening assays.

Many cellular analytical technologies measure only the average response from a cell population with an assumption that a clonal population is homogenous. The ensemble measurement often masks the difference among individual cells that can lead to misinterpretation. The advent of microfluidic technology has revolutionized single-cell analysis through precise manipulation of liquid and compartmentalizing single cells in small volumes (pico- to nano-liter). Due to its advantages from miniaturization, microfluidic systems offer an array of capabilities to study genomics, transcriptomics, and proteomics of a large number of individual cells. In this regard, microfluidic systems have emerged as a powerful technology to uncover cellular heterogeneity and expand the depth and breadth of single-cell analysis. This review will focus on recent developments of three microfluidic compartmentalization platforms (microvalve, microwell, and microdroplets) that target single-cell analysis spanning from proteomics to genomics. We also compare and contrast these three microfluidic platforms and discuss their respective advantages and disadvantages in single-cell analysis.

Shan Chen,1 Houming Liu,1 Tianpin Li,1 Wenjie Lai,1 Lei Liu,1 Youchun Xu,2 Jiuxin Qu1 1Department of Clinical Laboratory, Shenzhen Third People’s Hospital, National Clinical Research Center for Infectious Diseases, The Second Affiliated Hospital of Southern University of Science and Technology, Shenzhen, Guangdong Province, 518112, People’s Republic of China; 2Department of Biomedical Engineering, School of Medicine, Tsinghua University, Beijing, 100084, People’s Republic of ChinaCorrespondence: Jiuxin Qu, Department of Clinical Laboratory, Shenzhen Third People’s Hospital, National Clinical Research Center for Infectious Diseases, The Second Affiliated Hospital of Southern University of Science and Technology, Shenzhen, People’s Republic of China, Email Qujiuxin@163.com Youchun Xu, Department of Biomedical Engineering, School of Medicine, Tsinghua University, Beijing, People’s Republic of China, Email CYX040198@163.comBackground: The currently used conventional susceptibility testing for drug-resistant Mycobacterium tuberculosis (M.TB) is limited due to being time-consuming and having low efficiency. Herein, we propose the use of a microfluidic-based method to rapidly detect drug-resistant gene mutations using Kompetitive Allele-Specific PCR (KASP).Methods: A total of 300 clinical samples were collected, and DNA extraction was performed using the “isoChip®” Mycobacterium detection kit. Phenotypic susceptibility testing and Sanger sequencing were performed to sequence the PCR products. Allele-specific primers targeting 37 gene mutation sites were designed, and a microfluidic chip (KASP) was constructed using 112 reaction chambers to simultaneously detect multiple mutations. Chip validation was performed using clinical samples.Results: Phenotypic susceptibility of clinical isolates revealed 38 rifampicin (RIF)-resistant, 64 isoniazid (INH)-resistant, 48 streptomycin (SM)-resistant and 23 ethambutol (EMB)-resistant strains, as well as 33 multi-drug-resistant TB (MDR-TB) strains and 20 strains fully resistant to all four drugs. Optimization of the chip-based detection system for drug resistance detection showed satisfactory specificity and maximum fluorescence at a DNA concentration of 1× 101 copies/μL. Further analysis revealed that 76.32% of the RIF-resistant strains harbored rpoB gene mutations (sensitivity, 76.32%; specificity 100%), 60.93% of the INH-resistant strains had katG gene mutations (sensitivity, 60.93%; specificity, 100%), 66.66% of the SM-resistant strains carried drug resistance gene mutations (sensitivity, 66.66%; specificity, 99.2%), and 69.56% of the EMB-resistant strains had embB gene mutations (sensitivity, 69.56%; specificity, 100%). Further, the overall agreement between the microfluidic chip and Sanger sequencing was satisfactory, with a turnaround time of the microfluidic chip was approximately 2 hours, much shorter than the conventional DST method.Conclusion: The proposed microfluidic-based KASP assay provides a cost-effective and convenient method for detecting mutations associated with drug resistance in M. tuberculosis. It represents a promising alternative to the traditional DST method, with satisfactory sensitivity and specificity and a much shorter turnaround time.Graphical Abstract: Keywords: Mycobacterium tuberculosis, multidrug-resistance, drug resistance, resistance-conferring gene mutations, polymerase chain reaction, microfluidic analytical techniques, Sanger sequencing