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The abuse of antibiotics has become a global public safety issue, leading to the development of antimicrobial resistance (AMR). The development of antimicrobial susceptibility testing (AST) is crucial in reducing the growth of AMR. However, traditional AST methods are time-consuming (e.g., 24-72 h), labor-intensive, and costly. Here, we propose a controlled-diffusion centrifugal microfluidic platform (CCM) for rapid AST to obtain highly precise minimum inhibitory concentration (MIC) values. Antibiotic concentration gradients are generated by controlled moving and diffusing of antibiotic and buffer solution along the main microchannel within 3 min. The solution and bacterial suspension are then injected into the outermost reaction chamber by simple centrifugation. The CCM successfully determined the MIC for three commonly used antibiotics in clinical settings within 4-9 h. To further enhance practicality, reduce costs, and meet point-of-care testing demands, we have developed an integrated mobile detection platform for automated MIC value acquisition. The proposed CCM is a simple, low-cost, and portable method for rapid AST with broad clinical and in vitro applications., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

There remains an unmet need for a fully integrated microfluidic platform that can automatically perform multistep and multireagent immunoassays. Here, we proposed a novel online dual-active valve-based centrifugal microfluidic chip, termed DAVM, for fully automatic point-of-care immunoassay. Practically, the puncture valve, one of the dual active valves, is capable of achieving precise, on-demand, sequential release of prestored reagents, while the other valve-reversible active valve enables controlled retention and drainage of the reaction solutions. Thereby, our technology mitigates the challenges of hydrophilic/hydrophobic modifications and unstable valve control performance commonly observed in passive valve controls. As a proof of concept, the indirect enzymatic immunoblotting technique was employed on DAVM for fully automated immunological analysis of eight targets, yielding outcomes within an hour. Furthermore, we conducted a comparative analysis of 28 clinical samples with autoimmune diseases. According to 224 clinical data, the sample testing concordance rate between DAVM and the traditional instrument was 82%, with a target compliance rate of 97%. Therefore, our DAVM system has powerful potential for fully automated immunoassays.

Low-cost, rapid, and accurate acquisition of minimum inhibitory concentrations (MICs) is key to limiting the development of antimicrobial resistance (AMR). Until now, conventional antibiotic susceptibility testing (AST) methods are typically time-consuming, high-cost, and labor-intensive, making them difficult to accomplish this task. Herein, an electricity-free, portable, and robust handyfuge microfluidic chip was developed for on-site AST, termed handyfuge-AST. With simply handheld centrifugation, the bacterial-antibiotic mixtures with accurate antibiotic concentration gradients could be generated in less than 5 min. The accurate MIC values of single antibiotics (including ampicillin, kanamycin, and chloramphenicol) or their combinations against Escherichia coli could be obtained within 5 h. To further meet the growing demands of point-of-care testing, we upgraded our handyfuge-AST with a pH-based colorimetric strategy, enabling naked eye recognition or intelligent recognition with a homemade mobile app. Through a comparative study of 60 clinical data (10 clinical samples corresponding to six commonly used antibiotics), the accurate MICs by handyfuge-AST with 100% categorical agreements were achieved compared to clinical standard methods (area under curves, AUCs = 1.00). The handyfuge-AST could be used as a low-cost, portable, and robust point-of-care device to rapidly obtain accurate MIC values, which significantly limit the progress of AMR.

The frequent outbreak of global infectious diseases has prompted the development of rapid and effective diagnostic tools for the early screening of potential patients in point-of-care testing scenarios. With advances in mobile computing power and microfluidic technology, the smartphone-based mobile health platform has drawn significant attention from researchers developing point-of-care testing devices that integrate microfluidic optical detection with artificial intelligence analysis. In this article, we summarize recent progress in these mobile health platforms, including the aspects of microfluidic chips, imaging modalities, supporting components, and the development of software algorithms. We document the application of mobile health platforms in terms of the detection objects, including molecules, viruses, cells, and parasites. Finally, we discuss the prospects for future development of mobile health platforms., (© 2023. The Author(s).)

Point-of-care testing (POCT) has shown great advantages for public health monitoring in resource-limited settings. However, developing of POCT tools with automated and accurate quantitative dispensing of multiple reagents and samples is challenging. Here, we demonstrate a novel multi-reagents dispensing centrifugal microfluidics (MDCM) that allows rapid and automated dispensing of multiple reagents and samples with high throughput and accuracy. The MDCM was designed with multiple aliquoting units with the hydrophobic valve at different radial positions. All reagents and samples were loaded simultaneously, dispensed in parallel by centrifugation at low speed, and then introduced into the reaction chamber sequentially by centrifugation at high speed. Two MDCM chips are demonstrated, including a uniform concentration generator and a gradient concentration generator. The concentration coefficient of variation (CV) among the independent reaction chambers was lower than 0.56%, and the theoretical quantitative concentration gradient was strongly correlated with the actual concentration gradient (R 2  = 0.9938). We have successfully applied the MDCM to loop-mediated isothermal amplification (LAMP)-based nucleic acid detection for multiple infectious pathogens and antimicrobial susceptibility testing (AST) for kanamycin sulfate against E. coli. To further extend the applications, the MDCM has also been applied to bicinchoninic acid (BCA) protein assays with online calibration, reducing the detection time from 2 h to 10 min with a twenty-fold reduction in reagent consumption. These results indicated that the MDCM is a high potential platform for POCT., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The outbreak of global infectious diseases has posed a significant threat to public health, requiring the rapid and accurate diagnosis of pathogens promptly for the society to implement immediate control measures to prevent widespread pandemics. In this work, a magnet-actuated microfluidic array chip (MMAC) is developed with integrated sample processing and nucleic acid amplification for the rapid detection of multiple pathogens by loop-mediated isothermal amplification. In comparison to previous works, where fluid control was dependent on external equipment or finger-based manual pressing, the fluid control of the MMAC is realized by magnetically actuating a ferric oxide (Fe 3 O 4 ) doped polydimethylsiloxane (PDMS) layer that separates the sample from the LAMP reagent in a high-throughput manner, which not only reduces the complexity of fluid control but also enhances the repeatability of detection by eliminating variations in operation by different users. Examination with a testing sample containing Salmonella typhimurium and Escherichia coli showed high specificity for pathogen detection without cross-contamination. The lowest detection concentration was 5.2 copies per μL for Salmonella typhimurium with a detection time of 60 min. The proposed method demonstrated the simultaneous detection of multiple pathogens, which is potentially helpful in applications of immediate diagnosis.

Biomolecular phase separation is currently emerging in both the medical and life science fields. Meanwhile, the application of liquid-liquid phase separation has been extended to many fields including drug discovery, fibrous material fabrication, 3D printing, and polymer design. Although more than 8600 proteins and other synthetic macromolecules are capable of phase separation as recently reported, there is still a lack of a high-throughput approach to quantitatively characterize its phase behaviors. To meet this requirement, here, we proposed fast and high-resolution acquisition of biomolecular phase diagrams using microfluidic chips. Using this platform, we demonstrated the phase behavior of polyU/RRASLRRASLRRASL in a quantitative manner. Up to 1750 concentration conditions can be generated in 140 min. The detection limitation of our device to capture the saturation concentration for phase separation is about 5 times lower than that of the traditional turbidity method. Thus, our results provide a basis for the rapid acquisition of phase diagrams with high-throughput and pave the way for its wide application.

The integration of gel-based loop-mediated isothermal amplification (gLAMP) and finger-actuated microfluidic chip (μFAchip) was developed for the simultaneous detection of various different types of bacterial pathogens. The developed μFAchip consisted of three PDMS layers attached together by two adhesive tapes. Multiple chambers in the top PDMS layer were used for sample preparation, and the corresponding chambers in the bottom PDMS layer was used for long-term storage of LAMP reagents without DNA templates. The thin PDMS layer in the middle contained cross-shaped cuts as finger-actuated valves for fluid control. To reduce operation steps on the chip, such as pipetting and manipulation of samples, Whatman CloneSaver card was pre-embedded in the top chambers for on-chip DNA extraction and purification. Upon a simple press on the top layer, the finger-actuated valve was opened up, allowing DNA samples on the top layer flow into the bottom reaction chambers for gLAMP reaction. For POCT applications, on-chip LAMP reaction and imaging were conducted on a miniaturized peltier heater and a portable fluorescence imaging system respectively. Under the optimized condition, multiple pathogens were detected simultaneously with high selectivity and sensitivity (as low as 1.6 cells). The developed μFAchip provided a rapid and easy-to-operate platform for gLAMP-based pathogen detection, with the potential for in-field detection, especially in areas with limited resources., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Spatiotemporal manipulation of extracellular chemical environments with simultaneous monitoring of cellular responses plays an essential role in exploring fundamental biological processes and expands our understanding of underlying mechanisms. Despite the rapid progress and promising successes in manipulation strategies, many challenges remain due to the small size of cells and the rapid diffusion of chemical molecules. Fortunately, emerging microfluidic technology has become a powerful approach for precisely controlling the extracellular chemical microenvironment, which benefits from its integration capacity, automation, and high-throughput capability, as well as its high resolution down to submicron. Here, we summarize recent advances in microfluidics manipulation of the extracellular chemical microenvironment, including the following aspects: i) Spatial manipulation of chemical microenvironments realized by convection flow-, diffusion-, and droplet-based microfluidics, and surface chemical modification; ii) Temporal manipulation of chemical microenvironments enabled by flow switching/shifting, moving/flowing cells across laminar flows, integrated microvalves/pumps, and droplet manipulation; iii) Spatiotemporal manipulation of chemical microenvironments implemented by a coupling strategy and open-space microfluidics; and iv) High-throughput manipulation of chemical microenvironments. Finally, we briefly present typical applications of the above-mentioned technical advances in cell-based analyses including cell migration, cell signaling, cell differentiation, multicellular analysis, and drug screening. We further discuss the future improvement of microfluidics manipulation of extracellular chemical microenvironments to fulfill the needs of biological and biomedical research and applications., 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 © 2020 Elsevier B.V. All rights reserved.)

The circulating tumor cells (CTCs), originating from the primary tumor, play a vital role in cancer diagnosis, prognosis, disease monitoring, and precise therapy. However, the CTCs are extremely rare in the peripheral bloodstream and hard to be isolated. To overcome current limitations associated with CTC capture and analysis, the strategy incorporating nanostructures with microfluidic devices receives wide attention. Here, we demonstrated a three-dimensional microfluidic device (Rm-chip) for capturing cancer cells with high efficiency by integrating a novel hierarchical structure, the "Rhipsalis (Cactaceae)"-like micropillar array, into the Rm-chip. The PDMS micropillar array was fabricated by soft-lithography and rapid prototyping method, which was then conformally plated with a thin gold layer through electroless plating. EpCAM antibody was modified onto the surface of the micropillars through the thiol-oligonucleotide linkers in order to release captured cancer cells by DNase I treatment. The antibody-functionalized device achieved an average capture efficiency of 88% in PBS and 83.7% in whole blood samples. We believe the Rm-chip provided a convenient, economical, and versatile approach for cell analysis with wide potential applications.

The folding of G-quadruplex is hypothesized to undergo a complex process, from the formation of a hairpin structure to a triplex intermediate and to the final G-quadruplex. Currently, no experimental evidence has been found for the hairpin formation, because it folds in the time regime of 10-100 μs, entailing the development of microfluidic mixers with a mixing time of less than 10 μs. In this paper, we reported an ultrarapid micromixer with a mixing time of 5.5 μs, which represents the fastest turbulent micromixer to our best knowledge. Evaluations of the micromixer were conducted to confirm its mixing efficiency for small molecules and macromolecules. This new micromixer enabled us to interrogate the hairpin formation in the early folding process of human telomere G-quadruplex. The experimental kinetic evidence for the formation of hairpin was obtained for the first time.

Kinetic measurement of biomacromolecular interaction plays a significant role in revealing the underlying mechanisms of cellular activities. Due to the small diffusion coefficient of biomacromolecules, it is difficult to resolve the rapid kinetic process with traditional analytical methods such as stopped-flow or laminar mixers. Here, we demonstrated a unique continuous-flow laminar mixer based on microfluidic dual-hydrodynamic focusing to characterize the kinetics of DNA-protein interactions. The time window of this mixer for kinetics observation could cover from sub-milliseconds to seconds, which made it possible to capture the folding process with a wide dynamic range. Moreover, the sample consumption was remarkably reduced to <0.55 μL min⁻¹, over 1000-fold saving in comparison to those reported previously. We further interrogated the interaction kinetics of G-quadruplex and the single-stranded DNA binding protein, indicating that this novel micromixer would be a useful approach for analyzing the interaction kinetics of biomacromolecules.

Conventional neuronal analysis at the single neuron level usually involves culturing of neurons in vitro and analysis of neuronal activities by electrophysiological or pharmacological methods. However, the extracellular environments of in vitro neuronal analysis cannot mimic the exact surroundings of the neurons. Here, we report a microfluidic worm-chip for in vivo analysis of neuronal activities upon dynamic chemical stimulations. A comb-shaped microvalve was developed to immobilize whole animal for high-resolution imaging of neuronal activities. Using a sequential sample introduction system, multiple chemical stimuli were delivered to an individual Caenorhabditis elegans nose tip based on programmed interface shifting of laminar flows. ASH sensory neuron responses to various stimuli in individual C. elegans were quantitatively evaluated, and mutants were significantly defective in neuronal responses to certain stimulus in comparison to others. Sensory reduction in the magnitude of the response to repetitive chemical stimulation with different durations was also found. Our study explored the possibility of real-time detection of neuronal activities in individual animals upon multiple stimulations., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Caenorhabditis elegans (C. elegans) is a well-established model organism for investigating the correlations between behavioral and neuronal activities. Here, we demonstrated a microfluidic-based method that allowed stimulation-based neuronal analysis of immobilized C. elegans for identifying the neuronal effects of ethanol on the chemosensory responses of the right ASE (ASER) neuron. A one-piece microvalve was developed for the immobilization of C. elegans. Stimulations were realized by interface shifting of laminar flows. Well-fed transgenic worms expressing the calcium indicator G-CaMP in ASER neurons were used for in vivo fluorescence imaging. To evaluate the developed method, we first studied the effects of ethanol on the ASER neurons in response to a single NaCl stimulus. Results indicated that ethanol acutely suppressed the ON responses of ASER neurons to NaCl rather than the OFF response. Further studies of the adaptation of ASER neurons in response to NaCl and in the presence of ethanol suggested that ethanol interfered with the adaptation of neurons. The developed method exhibited the advantages of ease of operation and high throughput. We expect this new method to open up a new avenue for investigating the correlations between the behavioral and neuronal activities of C. elegans.

Microfluidic cell sorter allows efficient separation of small number of cells, which is beneficial in handling cells, especially primary cells that cannot be expanded to large populations. Here, we demonstrate a microfluidic fluorescence-activated cell sorter (muFACS) with a novel sorting mechanism, in which automatic on-chip sorting is realized by turning on/off the hydrodynamic gating valve when a fluorescent target is detected. Formation of the hydrodynamic gating valve was investigated by both numerical simulation and flow visualization experiment. Separation of fluorescent polystyrene beads was then conducted to evaluate this sorting mechanism and to optimize the separation conditions. Isolation of fluorescent HeLa-DsRed cells was further demonstrated with high purity and recovery rate. Viability of the sorted cells was also examined, suggesting a survival rate of more than 90%. We expect this sorting approach to find widespread applications in bioanalysis., (Copyright 2010 Elsevier B.V. All rights reserved.)

Chiral recognition of dansyl enantiomeric amino acids by microfluidic open tubular CEC (muOTCEC) with fluorescence detection was demonstrated. Avidin was employed as the chiral selector immobilized on the microchannel wall, which functioned as the chiral stationary phase (CSP) by physical adsorption. The condition of CSP on the glass wall was characterized using field emission SEM. Results indicated that avidin was homogenously distributed on the microchannel surface. Two parameters that played essential roles in muOTCEC for chiral recognition were investigated. Buffer pH could greatly change the amount of adsorption of avidin on the channel wall by altering the electrostatic attraction between them. Methanol, the organic additive to the running buffer, was also found significant for controlling the quality of the muOTCEC chiral separation by regulating the hydrophobic interaction between the enantiomers and the CSP. Under the optimized conditions, four dansyl racemic amino acids were then successfully separated by muOTCEC within 100 s with resolutions of 2.43, 1.88, 3.01 and 2.65 for dansyl-Ser, dansyl-Met, dansyl-Thr and dansyl-Val, respectively. Furthermore, a comparison with microfluidic CZE was investigated demonstrating that muOTCEC was a promising method for rapid chiral recognition.

Micro total analysis systems (microTAS) also referred to as "lab-on-a-chip" is one of the fastest progressing fields in biological and chemical analyses. In recent years, microTAS for single cell analysis has drawn the attention of researchers due to its significant advantages over traditional methods for single cell manipulation, fast cell sorting and integration of multiple functions. As the preliminary step for studying cells on chips, cell sorting using microfluidics have been investigated by researchers intensively. This article reviews the most recent advances on microfluidics-based cell sorting techniques including cell sorting principle, strategy, mechanism and procedure with emphases on the sorting mechanism and procedure. Furthermore, evaluation criteria for successful cell sorter are also discussed and future research directions are given.

[Display omitted] Microchip has long been studied and facilitated recent investigations in multiple biomedical and material fields. The advances in functional materials triggered several leaps in the development of microchip technology. Microarray chip, benefiting from micropatterning and nucleic acid nanotechnology, was firstly introduced around 1980 and rapidly facilitated genomics, proteomics, and biodetections. In the following generation, the microfluidic chips, raised from microelectromechanical systems (MEMS) and soft lithography, are revolutionizing several areas like biology, material fabrication, energy, and environmental science. More recently, the advances in materials fabrication keep expanding the frontiers of microchip platforms, like nanoscale fabrications and flexible device manufacturing. One of the most promising platforms is the wettability-patterned materials inspired by ubiquitous natural wetting creatures such as lotus leaf, spider silk, and Stenocara beetles. The unique property of handling liquids with no sophisticated equipment potentially facilitated the current microchip platform by combining the merits of microarray and microfluidics, and in turn, benefits material communities and beyond. In this featured article, we briefly introduce the state of art technologies to fabricate wettability-patterned chips and highlight some proof-of-concept demonstration of its emerging applications in material and biomedical science. We also give an outlook on its further developments including machine-learning micropattern manufacturing technology and reveal its potentiality to revolute several scientific areas. [ABSTRACT FROM AUTHOR]

Microfluidic technology has shown advantages of low sample consumption, reduced analysis time, high throughput, and potential for integration and automation. Coupling microfluidic chips to mass spectrometry (Chip-MS) can greatly improve the overall analytical performance of MS-based approaches and expand their potential applications. In this article, we review the advances of Chip-MS in the past decade, covering innovations in microchip fabrication, microchips coupled to electrospray ionization (ESI)-MS and matrix-assisted laser desorption/ionization (MALDI)-MS. Development of integrated microfluidic systems for automated MS analysis will be further documented, as well as recent applications of Chip-MS in proteomics, metabolomics, cell analysis, and clinical diagnosis. © 2014 Wiley Periodicals, Inc. Mass Spec Rev 34: 535-557, 2015. [ABSTRACT FROM AUTHOR]

Digital single-cell assays hold high potentials for the analysis of cell apoptosis and the evaluation of chemotherapeutic reagents for cancer therapy. In this paper, a microfluidic hydrodynamic trapping system was developed for digital single-cell assays with the capability of monitoring cellular dynamics over time. The microfluidic chip was designed with arrays of bypass structures for trapping individual cells without the need for surface modification, external electric force, or robotic equipment. After optimization of the bypass structure by both numerical simulations and experiments, a single-cell trapping efficiency of ∼90 % was achieved. We demonstrated the method as a digital single-cell assay for the evaluation of five clinically established chemotherapeutic reagents. As a result, the half maximal inhibitory concentration (IC) values of these compounds could be conveniently determined. We further modeled the gradual decrease of active drugs over time which was often observed in vivo after an injection to investigate cell apoptosis against chemotherapeutic reagents. The developed method provided a valuable means for cell apoptotic analysis and evaluation of anticancer drugs. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Intercellular Ca waves are propagation of Ca transients among cells that could be initiated by chemical stimulation. Current methods for analyzing intercellular Ca waves are difficult to realize localized chemical stimulations upon the target cell without interfering with adjacent contacting cells. In this paper, a simple and flexible microfluidic method was developed for investigating the intercellular communication of Ca signals. A cross-patterned microfluidic chip was designed and fabricated with polydimethylsiloxane as the structural material. Localized chemical stimulation was achieved by a new strategy based on hydrodynamic gating technique. Clusters of target cells were seeded at the location within 300 μm downstream of the intersection of the cross-shaped microchannel. Confined lateral molecular diffusion largely minimized the interference from diffusion-induced stimulation of adjacent cells. Localized stimulation of the target cell with adenosine 5′-triphosphate successfully induced the propagation of intercellular Ca waves among a population of adjacent contacting cells. Further inhibition studies verified that the propagation of calcium signals among NIH-3 T3 cells was dependent on direct cytosolic transfer via gap junctions. The developed microfluidic method provides a versatile platform for investigating the dynamics of intercellular communications. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

• Exosomes have been identified to hold exceptional value in clinical diagnostics and tumor therapy. • A short overview of conventional methods of exosome isolation is summarized. • The recent advancements of microfluidic strategy for exosomes isolation and detection are overviewed. • A brief overview of exosome-based drug delivery for tumor therapy is provided. • The current challenges and outlook of these fields are assessed. [Display omitted] Exosomes are a class of cell-secreted, nano-sized extracellular vesicles with a bilayer membrane structure of 30–150 nm in diameter. Their discovery and application have brought breakthroughs in numerous areas, such as liquid biopsies, cancer biology, drug delivery, immunotherapy, tissue repair, and cardiovascular diseases. Isolation of exosomes is the first step in exosome-related research and its applications. Standard benchtop exosome separation and sensing techniques are tedious and challenging, as they require large sample volumes, multi-step operations that are complex and time-consuming, requiring cumbersome and expensive instruments. In contrast, microfluidic platforms have the potential to overcome some of these limitations, owing to their high-precision processing, ability to handle liquids at a microscale, and integrability with various functional units, such as mixers, actuators, reactors, separators, and sensors. These platforms can optimize the detection process on a single device, representing a robust and versatile technique for exosome separation and sensing to attain high purity and high recovery rates with a short processing time. Herein, we overview microfluidic strategies for exosome isolation based on their hydrodynamic properties, size filtration, acoustic fields, immunoaffinity, and dielectrophoretic properties. We focus especially on advances in label-free isolation of exosomes with active biological properties and intact morphological structures. Further, we introduce microfluidic techniques for the detection of exosomal proteins and RNAs with high sensitivity, high specificity, and low detection limits. We summarize the biomedical applications of exosome-mediated therapeutic delivery targeting cancer cells. To highlight the advantages of microfluidic platforms, conventional techniques are included for comparison. Future challenges and prospects of microfluidics towards exosome isolation applications are also discussed. Although the use of exosomes in clinical applications still faces biological, technical, regulatory, and market challenges, in the foreseeable future, recent developments in microfluidic technologies are expected to pave the way for tailoring exosome-related applications in precision medicine. [ABSTRACT FROM AUTHOR]

In microbiological research, traditional methods for bacterial screening and antibiotic susceptibility testing are resource‐intensive. Microfluidics offers an efficient alternative with rapid results and minimal sample consumption, but the demand for cost‐effective, user‐friendly platforms persists in communities and hospitals. Inspired by the Magdeburg hemispheres, the strategy adapts to local conditions, leveraging omnipresent atmospheric pressure for self‐sealing of Rotation‐SlipChip (RSC) equipped with a 3D circular Christmas tree‐like microfluidic concentration gradient generator. This innovative approach provides an accessible and adaptable platform for microbiological research and testing in diverse settings. The RSC can avoid leakage concerns during multiple concentration gradient generation, chip‐rotating, and final long‐term incubation reaction (≥24 h). Furtherly, RSC subtypes adapted to different reactions can be fabricated in less than 15 min with cost less than $1, the result can be read through designated observational windows by naked‐eye. Moreover, the RSC demonstrates its capability for evaluating bacterial biomarker activity, enabling the rapid assessment of β‐galactosidase concentration and enzyme activity within 30 min, and the limit of detection can be reduced by 10‐fold. It also rapidly determines the minimum antibiotic inhibitory concentration and antibiotic combined medications results within 4 h. Overall, these low‐cost and user‐friendly RSC make them invaluable tools in determinations at previously impractical environment. [ABSTRACT FROM AUTHOR]

Public health events caused by pathogens have imposed significant economic and societal burdens. However, conventional methods still face challenges including complex operations, the need for trained operators, and sophisticated instruments. Here, we proposed a fully integrated and automated centrifugal microfluidic chip, also termed IACMC, for point-of-care multiplexed molecular diagnostics by harnessing the advantages of active and passive valves. The IACMC incorporates multiple essential components including a pneumatic balance module for sequential release of multiple reagents, a pneumatic centrifugation-assisted module for on-demand solution release, an on-chip silicon membrane module for nucleic acid extraction, a Coriolis force-mediated fluid switching module, and an amplification module. Numerical simulation and visual validation were employed to iterate and optimize the chip's structure. Upon sample loading, the chip automatically executes the entire process of bacterial sample lysis, nucleic acid capture, elution quantification, and isothermal LAMP amplification. By optimizing crucial parameters including centrifugation speed, direction of rotation, and silicone membrane thickness, the chip achieves exceptional sensitivity (twenty-five Salmonella or forty Escherichia coli) and specificity in detecting Escherichia coli and Salmonella within 40 min. The development of IACMC will drive advancements in centrifugal microfluidics for point-of-care testing and holds potential for broader applications in precision medicine including high-throughput biochemical analysis immune diagnostics, and drug susceptibility testing. • A novel fully integrated and automated centrifugal microfluidic chip, also termed IACMC, is proposed for point-of-care multiplexed molecular diagnostics by harnessing the advantages of active and passive valves. • Upon sample loading, the chip automatically executes the entire process of bacterial sample lysis, nucleic acid capture elution quantification, and isothermal LAMP amplification. • The IACMC achieves exceptional sensitivity (twenty-five Salmonella or forty Escherichia coli) and specificity in detecting Escherichia coli and Salmonella within 40 minutes. [ABSTRACT FROM AUTHOR]

Single cell analysis has aroused great interest with its remarkable ability to investigate cell-to-cell heterogeneity in large populations. However, probing protein information at single cells resolution including quantity, interactions, and dynamics have been great challenges as a result of the small size of cells, the complexity and a large concentration range of the protein and the lack of genome-wide amplification method. Fortunately, microfluidics capable of high throughput, high reproducibility, large parallelization, easy operability and low-cost, has been emerging as a powerful platform for the analysis of single cell proteins. In this review, we focus the recent advances of microfluidics in single cell resolution protein analysis, particularly covering the following aspects: (1) microfluidic electrophoresis (capillary electrophoresis and gel electrophoresis); (2) microfluidic cytometry (microfluidic flow cytometry, droplet based cytometry and image cytometry); (3) microfluidic array (micro wells, micro chambers, valve-based microfluidics and static droplet array microfluidics); (4) microfluidic probe and (5) microfluidics based mass spectrometry. • Microfluidic chip shows great promise in single cell resolution protein analysis field. • Recent advances in methods and technologies on microfluidic chip for single cell resolution protein analysis are reviewed. • Discusses the future improvements in microfluidics based single cell resolution protein analysis. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • A novel microfluidic device was proposed to achieve cell pairing at single cell level. • Highly efficient cell trapping (93%) and pairing (84%) were realized by using hydrodynamics, without external equipment. • The result showed that HUVEC could enhance the proliferation of HeLa. Abstract Understanding the mechanism of cell-cell communication is the key to unravel many physiological and pathological processes of multicellular organisms, but limited by lacking of highly efficient analytical approach, particularly at single cells resolution. In this work, we proposed a novel microfluidic-based method to achieve cell pairing for cell-cell communication investigation. Based on the only application of hydrodynamics, two types of cells were trapped by face-to-face heart-shaped micro-protrusions with high efficiency and single-cell accuracy. Oil-isolated micro-chambers were used to reduce cross-talk and spatial confinement on cells for long-term cell culture with high viability. The efficiency of 93% and 84% for single cells trapping and cells pairing were realized, respectively. To demonstrate the capability of the proposed platform for probing intercellular communication, the proliferation of cancer cell co-cultured with stromal cell was further discussed at single cells resolution. The result showed that the HUVEC could enhance the proliferation of HeLa cell. We believed that this microfluidic device as a flexible and reliable tool would have wide applications in the study of intercellular communication. [ABSTRACT FROM AUTHOR]

Cell signaling has been recognized as a highly dynamic process. How to capture such dynamics with changes of chemical microenvironment at single cell resolution among cells population without interfering with adjacent cells has been still an immense challenge. Here, we proposed a precision guided microfluidic chemical perfusion strategy for investigating chemical-activated cell signaling via dynamic imaging with high temporal (∼12 ms), spatial resolutions and precision. Theory for the microfluidic operation was first established and validated by both numerical simulations and experiments. A computer-aided multi-channel pressure control system was constructed to automatically implement chemical perturbations with various microperfusion times, relaxation times and number of cycles. The width and the position of the chemical microflow could also be precisely controlled with high reliability and accuracy. Investigations of intracellular and intercellular calcium signaling were successfully demonstrated using the proposed method. These results revealed that the precision guided microfluidic chemical perfusion strategy provided a versatile means for cell signaling analysis, which is potentially meaningful in chemical biology, cell biology and pharmacology. [ABSTRACT FROM AUTHOR]

Cell-based biosensor (CBB) has shown potential to provide a rapid and sensitive measurement means for monitoring specific biological substances. However, the detecting frequency and reusability remain a challenge for further application of CBB. In this paper, a microfluidic cell-based biosensor (μCBB) was developed, which consisted of a localized millisecond chemical pulse as sample introduction for pulsing individual cells without interfering with adjacent cells and single HeLa cells expressing endogenous P2Y receptors as the sensing elements. Gated injection of pinched flow was used to generate millisecond ATP pulses with predefined width. By integrating localized ATP pulse with predefined width, multiple sensing elements could be used to detect ATP pulses without impact on their functionality. Thus, the detecting frequency of the developed μCBB is multiplied by the number of available cells in comparison to that reported previously. We expect the developed method to be potentially useful to enhance the usability of CBB for biological and biomedical analysis. [ABSTRACT FROM AUTHOR]

In this work, we propose a microfluidic in-gel loop-mediated isothermal amplification (gLAMP) containing preloaded reagents as a self-contained microdevice for simultaneous detection of multiple pathogenic bacterial DNA in near point of care settings. The microchip consists of multiple channels with reaction chambers accommodating low-melting-point agarose and all LAMP reagents except the DNA samples, which can be kept at 4 °C for long-term storage. The current microchip enables simultaneous tests of four different bacterial DNA targets. After DNA samples were introduced into the microchip, gLAMP reactions were carried out on a hot plate at 65 °C. The gLAMP reaction conditions including the concentrations of the fluorescence indicator, Bst DNA polymerase, primers, and Mg 2+ ions were further optimized. Under the optimized conditions, detection of foodborne bacteria in serum samples, including Escherichia coli, Proteus hauseri, Vibrio parahaemolyticus, and Salmonella subsp. Enterica were demonstrated with high selectivity and sensitivity (as low as 3 copies/μL). The gLAMP microchip provides a simple and easy-to-operate platform for LAMP-based pathogen detection, which holds high potential for future applications in point-of-care settings. [ABSTRACT FROM AUTHOR]

In this paper, a new microfluidic approach, termed as gated pinched-flow (GPF), was developed for investigating cell-to-cell signaling dynamics coupling with optical imaging. It takes both advantages of conventional hydrodynamic gating and focusing, enabling precise on-chip chemical stimulation or perfusion upon targeted single cells among cells populations with high temporal (<50 ms) and spatial resolution. Theoretical model for GPF was first established and further validated by both numerical simulations and experiments. Stimulation of single HeLa cells was investigated to demonstrate the capability of the GPF for localized chemical stimulations of target single cells without interfering with adjacent cells. Guided by GPF, ATP-activated propagation of intercellular calcium waves (ICWs) among the seeded NIH-3T3 cells in the microchannel was then imaged with high repeatability. Inhibition investigations verified that those cell-to-cell calcium signals depended upon direct cytosolic transfer of molecules via gap junctions. The developed microfluidic method opens up a new avenue for cell-to-cell signaling studies and drug screening. [ABSTRACT FROM AUTHOR]

The Coronavirus disease 2019 (COVID-19) outbreak has urged the establishment of a global-wide rapid diagnostic system. Current widely-used tests for COVID-19 include nucleic acid assays, immunoassays, and radiological imaging. Immunoassays play an irreplaceable role in rapidly diagnosing COVID-19 and monitoring the patients for the assessment of their severity, risks of the immune storm, and prediction of treatment outcomes. Despite of the enormous needs for immunoassays, the widespread use of traditional immunoassay platforms is still limited by high cost and low automation, which are currently not suitable for point-of-care tests (POCTs). Microfluidic chips with the features of low consumption, high throughput, and integration, provide the potential to enable immunoassays for POCTs, especially in remote areas. Meanwhile, luminescence detection can be merged with immunoassays on microfluidic platforms for their good performance in quantification, sensitivity, and specificity. This review introduces both homogenous and heterogenous luminescence immunoassays with various microfluidic platforms. We also summarize the strengths and weaknesses of the categorized methods, highlighting their recent typical progress. Additionally, different microfluidic platforms are described for comparison. The latest advances in combining luminescence immunoassays with microfluidic platforms for POCTs of COVID-19 are further explained with antigens, antibodies, and related cytokines. Finally, challenges and future perspectives were discussed. • Immunological signatures of the life cycles of COVID-19 are introduced. • Luminescence immunoassays on microfluidic chips for point-of-care (POC) diagnosis of COVID-19 are well detailed. • Existent challenges and future perspectives for POC immunoassays are further discussed. [ABSTRACT FROM AUTHOR]

Abstract: The use of cell-based biosensors is usually limited by agonist-induced desensitization of cell-surface receptors. In this work, a microfluidic cell-based biosensor (μCBB) was developed for the detection of ATP in liquid environments. It consists of a millisecond chemical pulse generator for sample introduction in a pulsatile manner and a single NIH-3T3 cell expressing endogenous P2Y receptors as the sensing element. ATP solutions were used to simulate input signals for investigating the μCBB. By controlling negative pressures on two outlets of a cross-shaped microfluidic chip, pulses of ATP solutions were generated based on hydrodynamic gated injection. With ATP pulses of 100ms every 50s, the amplitude of the resulting calcium spikes maintained at a similar level, suggesting that the receptor desensitization was minimized. Consequently, the developed μCBB could be used for detecting pulsatile samples with extended use times. The sensitivity of the μCBB for detecting ATP was further determined and the cellular responses to millisecond ATP pulses were investigated in comparison to long-term stimulations. [Copyright &y& Elsevier]

• A low-cost and rapid agarose-based microfluidic chip for exosomes preconcentration and detection was proposed. • Detection sensitivity of a LOD of ∼103 exosomes along with the extremely low volume of samples (as low as 1 μL). • IIn situ concentration of different cell line's exosomes and detection of exosomal microRNA simultaneously. Exosomes, together with their accompanying proteins and nucleic acids have been increasingly recognized as biomarkers for non-invasive tumor diagnostic and prognostic. However, rapid and efficient detection of exosomes remains challenging, due to their small size (30−150 nm), and low concentration. Thus, to guarantee, especially when cancer screening requires detection limits lower than exosome concentrations, it not only needs to isolate and purify exosomes, but it needs to preconcentrate them as well. Here, a low-cost, rapid, and portable agarose-based microfluidic chip for exosome concentration and in situ exosomal microRNA detection was developed, termed isExoCD (in situ exosome concentration and detection). The target exosomes loaded at the entrance will be automatically enriched at the closed end of the microchannel by leveraging the capillary effect and the strong water permeability of the agarose gel. To detect the exosomal microRNA, we integrated the catalyzed hairpin assembly (CHA) based strategy into the isExoCD, allowing high sensitive detection of exosomes with a limit of detection (LOD) of ∼103. Using our method, we can successfully distinguish cancer cell-derived exosomes from other exosomes that obtained other cell types. Thus, our platform paves a new avenue to early cancer detection, liquid biopsy, and point-of-care diagnosis. [ABSTRACT FROM AUTHOR]