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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]

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.

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]

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.

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 utilization of microfluidic analysis technology has resulted in the advancement of fast pathogenic bacteria detection, which can accurately provide information on biochemical reactions in a single cell and enhance detection efficiency. Nevertheless, the achievement of rapid and effective in situ detection of single-bacteria arrays remains a challenge due to the complexity of bacterial populations and low Reynolds coefficient fluid, resulting in insufficient diffusion. We develop microwell droplet array chips from the lateral hydrodynamic wetting approach to address this issue. The sidewall of the microwell gradually opens which aids in advancing the liquid–air interface and facilitates the impregnation of the solid microwells, preserving the Wenzel state and assisting in resisting the liquid force to separation from the drop. The feasibility of preparing cell arrays and identifying them inside the microwells was demonstrated through the simulated streamlined distribution of gradual and traditional microwells with different sizes. The water-based ink diffusion experiment examined the relationship between diffusion efficiency and flow velocity, as well as the position of the microwell relative to the channel. It showed that the smaller gradual microwell still has a good diffusion efficiency rate at a flow velocity of 2.1 μL min−1 and that the infiltration state is easier to adjust. With this platform, we successfully isolated a mixed population containing E. coli and S. aureus, obtained single-bacteria arrays, and performed Gram assays after in situ propagation. After 20 hours of culture, single bacteria reproduced demonstrating the capability of this platform to isolate, cultivate, and detect pathogenic bacteria. [ABSTRACT FROM AUTHOR]

Flow cytometry is an essential technique in single particle analysis and cell sorting for further downstream diagnosis, exhibiting high-throughput and multiplexing capabilities for many biological and biomedical applications. Although many hydrodynamic focusing-based microfluidic cytometers have been demonstrated with reduced size and cost to adapt to point-of-care settings, the operating conditions are not characterized systematically. This study presents the flow transition process in the hydrodynamic focusing mechanism when the flow rate or the Reynolds number increases. The characteristics of flow fields and mass transport were studied under various operating conditions, including flow rates and microchannel heights. A transition from the squeezed focusing state to the over-squeezed anti-focusing state in the hydrodynamic focusing regime was observed when the Reynolds number increased above 30. Parametric studies illustrated that the focusing width increased with the Reynolds number but decreased with the microchannel height in the over-squeezed state. The microfluidic cytometric analyses using microbeads and E. coli show that the recovery rate was maintained by limiting the Reynolds number to 30. The detailed analysis of the flow transition will provide new insight into microfluidic cytometric analyses with a broad range of applications in food safety, water monitoring and healthcare sectors. [ABSTRACT FROM AUTHOR]

As core parts of microfluidic chip analysis systems, micromixers show robust applications in wide fields. However, restricted by the fabrication technology, it remains challenging to achieve high-quality micromixers with both delicately designed structure and efficient mixing. In this study, based on the theory of chaotic mixing, sinusoidal structures with variable phases were designed and then fabricated through scanning probe lithography (SPL) and post-selective etching. It was found that scratches with phase differences can lead to the periodic formation of amorphous silicon (a-Si), which can resist etching. Consequentially, misaligned sine channels with thick–thin alternating 3D shapes can be generated in situ from the scratched traces after the etching. Further analysis showed that a thicker a-Si layer can be obtained by reducing the line spacing in the scratching, confirmed by Raman detections and simulations. With the proposed method, the misaligned sine micromixer was achieved with higher mixing efficiency than ever. The duplicating process was also investigated for high-precision production of micromixers. The study provided strategies for the miniaturization of high-performance microfluidic chips. [ABSTRACT FROM AUTHOR]

Dispensing micron-scale droplets from a suspended nozzle is important for applications in bioprinting, analytical chemistry, and pharmaceutical formulation. Here, we describe a general approach to eject droplets from microfluidic devices using superhydrophobic patterning; this facilitates release of wetted fluids, allowing droplets to break contact with channel surfaces and travel along regular paths to achieve a printing accuracy of ∼3 μm. We demonstrate the utility of the approach by using it to print droplets of varied composition from a microfluidic mixing device. Our approach is compatible with common fabrication techniques making it applicable to devices configured for diverse applications.

Infusion pumps have been widely used in clinical settings for the administration of medications and fluids. We present the digital droplet infusion (DDI) device, a low-cost, high-precision digital infusion system, utilizing a microfluidic discretization unit to convert continuous flow into precisely delivered droplet aliquots and a valving unit to control the duration and frequency of flow discretization. The DDI device relies on a distinct capillarity-dominated process of coalescence and pinch-off of droplets for flow digitization, which is monitored by a pair of conductive electrodes located before and after the junction. The digital feedback-controlled flow rate can be employed to adjust a solenoid valve for refined infusion management. With this unique digital microfluidic approach, the DDI technology enables a simple yet powerful infusion system with an ultrahigh resolution of digital droplet transfer volume, as small as 57 nL, which is three orders of magnitude lower than that of clinical standard infusion pumps, as well as a wide range of digitally adjustable infusion rates ranging from 0.1 mL h-1 to 10 mL h-1, in addition to an array of programmable infusion profiles and safety features. Its modular design enables fast assembly using only off-the-shelf and 3D-printed components. Overall, benefiting from its simple device architecture and excellent infusion performance, the DDI technology has great potential to become the next-generation clinical standard for drug delivery with its high precision and ultimate portability at a low cost.

Droplet-based microfluidic technology is a powerful tool for generating large numbers of monodispersed nanoliter-sized droplets for ultra-high throughput screening of molecules or single cells. Yet further progress in the development of methods for the real-time detection and measurement of passing droplets is needed for achieving fully automated systems and ultimately scalability. Existing droplet monitoring technologies are either difficult to implement by non-experts or require complex experimentation setups. Moreover, commercially available monitoring equipment is expensive and therefore limited to a few laboratories worldwide. In this work, we validated for the first time an easy-to-use, open-source Bonsai visual programming language to accurately measure in real-time droplets generated in a microfluidic device. With this method, droplets are found and characterized from bright-field images with high processing speed. We used off-the-shelf components to achieve an optical system that allows sensitive image-based, label-free, and cost-effective monitoring. As a test of its use we present the results, in terms of droplet radius, circulation speed and production frequency, of our method and compared its performance with that of the widely-used ImageJ software. Moreover, we show that similar results are obtained regardless of the degree of expertise. Finally, our goal is to provide a robust, simple to integrate, and user-friendly tool for monitoring droplets, capable of helping researchers to get started in the laboratory immediately, even without programming experience, enabling analysis and reporting of droplet data in real-time and closed-loop experiments. [ABSTRACT FROM AUTHOR]

Microfluidic-based lab-on-a-chip technology is a multidisciplinary approach, which has evolved rapidly in the past decade and remains a hot research topic as a promising microanalysis platform for a plethora of biomedical applications. Microfluidic chips have been successfully applied in cancer diagnosis and monitoring, given that they can lead to the effective separation and analysis of cancer-derived substances such as extracellular vesicles (EVs), circulating tumour cells (CTCs) and circulating DNA (ctDNA), proteins and other metabolites. In particular, EVs and CTCs are two outstanding objects for cancer liquid biopsy, which share similar membrane structures but possess different sizes. Through molecular typing and concentration detection of EVs, CTCs and ctDNA, disease-related information can be well-learned, including the development stage and prognosis of cancer. However, the conventional separation and detection methods are often time-consuming with limited efficiency. In comparison, the use of microfluidic platforms can effectively simplify the separation and enrichment process and improve the detection efficiency significantly. Although review papers have been published on the application of microfluidic chips for the analysis of objects of liquid biopsy, generally they focused on a specific detection target, lacking a descriptive extraction of the commonality of LOC devices used in liquid biopsy. Thus, few of them present a comprehensive overview and outlook on the design and application of microfluidic chips for liquid biopsy. This motivated us to prepare this review paper, which is divided into 4 parts. The first part aims to elucidate the material selection and fabrication approaches of microfluidic chips. In the second part, the important separation strategies, including physical methods and biological methods, are discussed. The third part highlights the advanced on-chip technologies for the detection of EVs, CTCs and ctDNA by providing practical examples. In the fourth part, novel on-chip applications of single cells/exosomes are introduced. Finally, the prospective outlook and challenges for the long-term development of on-chip assays are envisioned and discussed. [ABSTRACT FROM AUTHOR]

Current encapsulation approaches control the number of particles encapsulated per droplet, but not the particle types; consequently, they are unable to generate droplets containing combinations of distinct particle types, limiting the reactions that can be performed. We describe a microfluidic particle zipper that allows the number and types of particles encapsulated in every droplet to be controlled. The approach exploits self-ordering to generate repeating particle patterns that allow controlled encapsulation in droplets. We use the method to combine barcode particles with gel encapsulated cells to profile multiple disease relevant genomic loci with single cell sequencing. Particle zippers can operate in series to generate complex particle compositions in droplets.

Sepsis due to antimicrobial resistant pathogens is a major health problem worldwide. The inability to rapidly detect and thus treat bacteria with appropriate agents in the early stages of infections leads to excess morbidity, mortality, and healthcare costs. Here we report a rapid diagnostic platform that integrates a novel one-step blood droplet digital PCR assay and a high throughput 3D particle counter system with potential to perform bacterial identification and antibiotic susceptibility profiling directly from whole blood specimens, without requiring culture and sample processing steps. Using CTX-M-9 family ESBLs as a model system, we demonstrated that our technology can simultaneously achieve unprecedented high sensitivity (10 CFU per ml) and rapid sample-to-answer assay time (one hour). In head-to-head studies, by contrast, real time PCR and BioRad ddPCR only exhibited a limit of detection of 1000 CFU per ml and 50-100 CFU per ml, respectively. In a blinded test inoculating clinical isolates into whole blood, we demonstrated 100% sensitivity and specificity in identifying pathogens carrying a particular resistance gene. We further demonstrated that our technology can be broadly applicable for targeted detection of a wide range of antibiotic resistant genes found in both Gram-positive (vanA, nuc, and mecA) and Gram-negative bacteria, including ESBLs (blaCTX-M-1 and blaCTX-M-2 families) and CREs (blaOXA-48 and blaKPC), as well as bacterial speciation (E. coli and Klebsiella spp.) and pan-bacterial detection, without requiring blood culture or sample processing. Our rapid diagnostic technology holds great potential in directing early, appropriate therapy and improved antibiotic stewardship in combating bloodstream infections and antibiotic resistance.

Microdroplet screening of microorganisms can improve the rate of strain selection and characterization within the canonical design–build–test paradigm. However, a full analysis of the microdroplet environment and how well these conditions translate to culturing conditions and techniques is lacking in the field. Quantification of three different biosensor/analyte combinations at 12 hour timepoints reveals the potential for extended dose–response ranges as compared to traditional in vitro conditions. Using these dynamics, we present an application and analysis of microfluidic droplet screening utilizing whole-cell biosensors, ultimately identifying an altered productivity profile of itaconic acid in a Yarrowia lipolytica-based piggyBac transposon library. Specifically, we demonstrate that the timepoint for microdroplet selection can influence the outcome of the selection and thus shift the identified strain productivity and final titer. In this case, strains selected at earlier timepoints showed increased early productivity in flask scale, with the converse true as well. Differences in response indicate microdroplet assays require tailored development to more accurately sort for phenotypes that are scalable to larger incubation volumes. Likewise, these results further highlight that screening conditions are critical parameters for success in high-throughput applications. [ABSTRACT FROM AUTHOR]

Microfluidic chambers are powerful tools for studying axonal mRNA localization and translation in neurons. In addition to specific manipulation and measurements of axons, microfluidic chambers are used for collecting axonal materials to perform axonal transcriptome analysis. However, traditional bipartite and tripartite chambers have limitations either in purity or quantity of collected axons. Here, we improved the design of traditional chambers. Moreover, we developed two new quantitative chambers, multi-compartmental quantitative bipartite chamber (MQBC) and long quantitative tripartite chamber (LQTC). Compared with the traditional chambers, MQBC and LQTC could dramatically increase the efficiency in collecting axonal RNA. Finally, we applied these chambers to do comparative axon transcriptome analysis of different types of neurons. Thus, our newly designed quantitative chambers significantly improve axon collection efficiency and facilitate axonal transcriptome analysis. [ABSTRACT FROM AUTHOR]

Adoptive T cell transfer, in particular TCR T cell therapy, holds great promise for cancer immunotherapy with encouraging clinical results. However, finding the right TCR T cell clone is a tedious, time-consuming, and costly process. Thus, there is a critical need for single cell technologies to conduct fast and multiplexed functional analyses followed by recovery of the clone of interest. Here, we use droplet microfluidics for functional screening and real-time monitoring of single TCR T cell activation upon recognition of target tumor cells. Notably, our platform includes a tracking system for each clone as well as a sorting procedure with 100% specificity validated by downstream single cell reverse-transcription PCR and sequencing of TCR chains. Our TCR screening prototype will facilitate immunotherapeutic screening and development of T cell therapies.

From primary tumours and disseminating to secondary organs, cancer cells experience a wide variety of fluid flow profiles when passing through blood vessels or the lymphatic system before extravasation. Sinusoidal capillaries are a common site for extravasation. Therefore, we aim to investigate how metastatic cancer cells react to a biophysical cue such as capillary fluid flow by quantifying its effect on metastatic cell cycle progression, motility, cell and nuclear volume, and morphology. We use MDA-MB-231 breast cancer cells genetically modified with fluorescent ubiquitination-based cell cycle indicator 2 (FUCCI2) as a model system. Single cells are trapped using a microfluidic device and exposed to different laminar flows. Quantitative time-lapse imaging in both 2D epifluorescence and 3D confocal microscopy is performed using in-house software FUCCItrack. In addition, 3D time-lapse with cell and nuclear segmentation is performed with a deep learning approach to streamline the image processing of big datasets. We show that at a single cell level, faster fluid flow leads to a shorter S/G2/M phase and an overall shorter cell cycle, as well as increase in cell motility irrespective of the flow direction. 3D time-lapse confocal imaging of MDA-FUCCI2 single cells reveals the evolution of cell and nuclear volume and morphology as a function of a specific cell cycle phase. Both cell and nuclear volume increase linearly over time. Cell morphology elongates more strongly during the S/G2/M phase, whereas the nuclear shape remains constant. Under the highest flow conditions, only during the S/G2/M phase can we observe a more elongated nucleus, while the cell sphericity remains similar to the control. Collectively, this data, together with the deep learning approach, provides new insights into the potential impact of fluid flow at a single cell level. [ABSTRACT FROM AUTHOR]

Lab-on-a-chip (LOC) applications have emerged as invaluable physical and life sciences tools. The advantages stem from advanced system miniaturization, thus, requiring far less sample volume while allowing for complex functionality, increased reproducibility, and high throughput. However, LOC applications necessitate extensive sensor miniaturization to leverage these inherent advantages fully. Atom-sized quantum sensors are highly promising to bridge this gap and have enabled measurements of temperature, electric and magnetic fields on the nano- to microscale. Nevertheless, the technical complexity of both disciplines has so far impeded an uncompromising combination of LOC systems and quantum sensors. Here, we present a fully integrated microfluidic platform for solid-state spin quantum sensors, like the nitrogen-vacancy (NV) center in diamond. Our platform fulfills all technical requirements, such as fast spin manipulation, enabling full quantum sensing capabilities, biocompatibility, and easy adaptability to arbitrary channel and chip geometries. To illustrate the vast potential of quantum sensors in LOC systems, we demonstrate various NV center-based sensing modalities for chemical analysis in our microfluidic platform, ranging from paramagnetic ion detection to high-resolution microscale NV-NMR. Consequently, our work opens the door for novel chemical analysis capabilities within LOC devices with applications in electrochemistry, high-throughput reaction screening, bioanalytics, organ-on-a-chip, or single-cell studies. [ABSTRACT FROM AUTHOR]

Controllable mass production of monodisperse droplets plays a key role in numerous fields ranging from scientific research to industrial application. Microfluidic ladder networks show great potential in mass production of monodisperse droplets, but their design with uniform microflow distribution remains challenging due to the lack of a rational design strategy. Here an effective design strategy based on backstepping microflow analysis (BMA) is proposed for the rational development of microfluidic ladder networks for mass production of controllable monodisperse microdroplets. The performance of our BMA rule for rational microfluidic ladder network design is demonstrated by using an existing analogism-derived rule that is widely used for the design of microfluidic ladder networks as the control group. The microfluidic ladder network designed by the BMA rule shows a more uniform flow distribution in each branch microchannel than that designed by the existing rule, as confirmed by single-phase flow simulation. Meanwhile, the microfluidic ladder network designed by the BMA rule allows mass production of droplets with higher size monodispersity in a wider window of flow rates and mass production of polymeric microspheres from such highly monodisperse droplet templates. The proposed BMA rule provides new insights into the microflow distribution behaviors in microfluidic ladder networks based on backstepping microflow analysis and provides a rational guideline for the efficient development of microfluidic ladder networks with uniform flow distribution for mass production of highly monodisperse droplets. Moreover, the BMA method provides a general analysis strategy for microfluidic networks with parallel multiple microchannels for rational scale-up. [ABSTRACT FROM AUTHOR]

The rapid screening and isolation of single leukemia cells from blood has become critical for early leukemia detection and tumor heterogeneity interrogation. However, due to the size overlap between leukemia cells and the more abundant white blood cells (WBCs), the isolation and identification of leukemia cells individually from peripheral blood is extremely challenging and often requires immunolabeling or cytogenetic assays. Here we present a rapid and label-free single leukemia cell identification platform that combines: (1) high-throughput size-based separation of hemocytes via a single-cell trapping array, and (2) leukemia cell identification through phasor approach and fluorescence lifetime imaging microscopy (phasor-FLIM), to quantify changes between free/bound nicotinamide adenine dinucleotide (NADH) as an indirect measurement of metabolic alteration in living cells. The microfluidic trapping array designed with 1600 highly-packed addressable single-cell traps can simultaneously filter out red blood cells (RBCs) and trap WBCs/leukemia cells, and is compatible with low-magnification imaging and fast-speed fluorescence screening. The trapped single leukemia cells, e.g., THP-1, Jurkat and K562 cells, are distinguished from WBCs in the phasor-FLIM lifetime map, as they exhibit significant shift towards shorter fluorescence lifetime and a higher ratio of free/bound NADH compared to WBCs, because of their glycolysis-dominant metabolism for rapid proliferation. Based on a multiparametric scheme comparing the eight parameter-spectra of the phasor-FLIM signatures, spiked leukemia cells are quantitatively distinguished from normal WBCs with an area-under-the-curve (AUC) value of 1.00. Different leukemia cell lines are also quantitatively distinguished from each other with AUC values higher than 0.95, demonstrating high sensitivity and specificity for single cell analysis. The presented platform is the first to enable high-density size-based single-cell trapping simultaneously with RBC filtering and rapid label-free individual-leukemia-cell screening through non-invasive metabolic imaging. Compared to conventional biomolecular diagnostics techniques, phasor-FLIM based single-cell screening is label-free, cell-friendly, robust, and has the potential to screen blood in clinical volumes through parallelization.

Microfluidic droplet sorting allows selection of subpopulations of cells, nucleic acids, and biomolecules with soluble assays. Dielectrophoresis is widely used for sorting because it generates strong forces on droplets, actuates rapidly, and is easy to integrate into microfluidic chips. However, existing device designs apply a short force, limiting the deflection of droplets, and therefore the speed and reliability of sorting. We describe a concentric design that applies a long force, allowing large deflections and increased reliability. We demonstrate the utility of this design by sorting polydisperse emulsions, which are typically difficult to sort with high purity.

Microfluidic droplet sorting allows selection of subpopulations of cells, nucleic acids, and biomolecules with soluble assays. Dielectrophoresis is widely used for sorting because it generates strong forces on droplets, actuates rapidly, and is easy to integrate into microfluidic chips. However, existing device designs apply a short force, limiting the deflection of droplets, and therefore the speed and reliability of sorting. We describe a concentric design that applies a long force, allowing large deflections and increased reliability. We demonstrate the utility of this design by sorting polydisperse emulsions, which are typically difficult to sort with high purity.

The invasion of malignant cells into tissue is a critical step in the progression of cancer. While it is increasingly appreciated that cells within a tumor differ in their invasive potential, it remains nearly unknown how these differences relate to cell-to-cell variations in protein expression. Here, we introduce a microfluidic platform that integrates measurements of invasive motility and protein expression for single cells, which we use to scrutinize human glioblastoma tumor-initiating cells (TICs). Our live-cell imaging microdevice is comprised of polyacrylamide microchannels that exhibit tissue-like stiffness and present chemokine gradients along each channel. Due to intrinsic differences in motility, cell subpopulations separate along the channel axis. The separated cells are then lysed in situ and each single-cell lysate is subjected to western blotting in the surrounding polyacrylamide matrix. We observe correlations between motility and Nestin and EphA2 expression. We identify protein-protein correlations within single TICs, which would be obscured with population-based assays. The integration of motility traits with single-cell protein analysis - on the same cell - offers a new means to identify druggable targets of invasive capacity.

Cell-matrix and cell-cell interactions influence intracellular signalling and play an important role in physiologic and pathologic processes. Detachment of cells from the surrounding microenvironment alters intracellular signalling. Here, we demonstrate and characterise an integrated microfluidic device to culture single and clustered cells in tuneable microenvironments and then directly analyse the lysate of each cell in situ, thereby eliminating the need to detach cells prior to analysis. First, we utilise microcontact printing to pattern cells in confined geometries. We then utilise a microscale isoelectric focusing (IEF) module to separate, detect, and analyse lamin A/C from substrate-adhered cells seeded and cultured at varying (500, 2000, and 9000 cells per cm2) densities. We report separation performance (minimum resolvable pI difference of 0.11) that is on par with capillary IEF and independent of cell density. Moreover, we map lamin A/C and β-tubulin protein expression to morphometric information (cell area, circumference, eccentricity, form factor, and cell area factor) of single cells and observe poor correlation with each of these parameters. By eliminating the need for cell detachment from substrates, we enhance detection of cell receptor proteins (CD44 and β-integrin) and dynamic phosphorylation events (pMLCS19) that are rendered undetectable or disrupted by enzymatic treatments. Finally, we optimise protein solubilisation and separation performance by tuning lysis and electrofocusing (EF) durations. We observe enhanced separation performance (decreased peak width) with longer EF durations by 25.1% and improved protein solubilisation with longer lysis durations. Overall, the combination of morphometric analyses of substrate-adhered cells, with minimised handling, will yield important insights into our understanding of adhesion-mediated signalling processes.

Despite the increasing importance of positron emission tomography (PET) imaging in research and clinical management of disease, access to myriad new radioactive tracers is severely limited due to their short half-lives (which requires daily production) and the high cost and complexity of tracer production. The application of droplet microfluidics based on electrowetting-on-dielectric (EWOD) to the field of radiochemistry can significantly reduce the amount of radiation shielding necessary for safety and the amount of precursor and other reagents needed for the synthesis. Furthermore, significant improvements in the molar activity of the tracers have been observed. However, widespread use of this technology is currently hindered in part by the high cost of prototype chips and the operating complexity. To address these issues, we developed a novel microfluidic device based on patterned wettability for multi-step radiochemical reactions in microliter droplets and implemented automated systems for reagent loading and collection of the crude product after synthesis. In this paper, we describe a simple and inexpensive method for fabricating the chips, demonstrate the feasibility of prototype chips for performing multi-step radiochemical reactions to produce the PET tracers [18F]fallypride and [18F]FDG, and further show that synthesized [18F]fallypride can be used for in vivo mouse imaging.

Three-dimensional pluripotent stem cell (PSC) cultures have the ability to undergo differentiation, self-organization, and morphogenesis to yield complex, in vitro tissue models that recapitulate key elements of native tissues. These tissue models offer a system for studying mechanisms of tissue development, investigating disease mechanisms, and performing drug screening. It remains challenging, however, to standardize PSC aggregate differentiation and morphogenesis methods due to heterogeneity stemming from biological and environmental sources. It is also difficult to monitor and assess large numbers of individual samples longitudinally throughout culture using typical batch-based culture methods. To address these challenges, we have developed a microfluidic platform for culture, longitudinal monitoring, and phenotypic analysis of individual stem cell aggregates. This platform uses a hydrodynamic loading principle to capture pre-formed stem cell aggregates in independent traps. We demonstrated that multi-day culture of aggregates in this platform reduces heterogeneity in phenotypic parameters such as size and morphology. Additionally, we showed that culture and analysis steps can be performed sequentially in the same platform, enabling correlation of multiple modes of analysis for individual samples. We anticipate this platform being applied to improve abilities for phenotypic analysis of PSC aggregate tissues and to facilitate research in standardizing culture systems in order to dually increase the yield and reduce the heterogeneity of PSC-derived tissues.

Synthetic biology is an interdisciplinary field that aims to engineer biological systems for useful purposes. Organism engineering often requires the optimization of individual genes and/or entire biological pathways (consisting of multiple genes). Advances in DNA sequencing and synthesis have recently begun to enable the possibility of evaluating thousands of gene variants and hundreds of thousands of gene combinations. However, such large-scale optimization experiments remain cost-prohibitive to researchers following traditional molecular biology practices, which are frequently labor-intensive and suffer from poor reproducibility. Liquid handling robotics may reduce labor and improve reproducibility, but are themselves expensive and thus inaccessible to most researchers. Microfluidic platforms offer a lower entry price point alternative to robotics, and maintain high throughput and reproducibility while further reducing operating costs through diminished reagent volume requirements. Droplet microfluidics have shown exceptional promise for synthetic biology experiments, including DNA assembly, transformation/transfection, culturing, cell sorting, phenotypic assays, artificial cells and genetic circuits.

Advanced biomedical device coatings have shown significant promise in delivery of therapeutics (e.g., small-molecule drugs, proteins) for a wide range of medical interventions ranging from targeted cancer therapy to management of atherosclerosis. In order to accelerate the development of such coatings, there is a need for tools to investigate the loading capacity and release kinetics with high temporal resolution and in a variety of physiological conditions. To address this need, we report a microfluidic platform, where the coating on a substrate can be mounted onto the microchannel and the device can be configured in two physiologically-relevant modes: (i) flow-mode allows for monitoring the release from the coating in contact with a liquid flowing at a specific rate, modeling the case of a drug-eluting stent. (ii) Static-mode, where the channel is filled with a stationary gel, mimics the case of drug-eluting brain implant. We demonstrate the utility of the platform with a fluorescein-loaded nanoporous gold coating and monitor in real-time the release kinetics both under deionized water infusion and an agarose gel-filled channel via fluorescence microscopy coupled to a LabVIEW-based interface.

The ability to harvest single cells from tissues is currently a bottleneck for cell-based diagnostic technologies, and remains crucial in the fields of tissue engineering and regenerative medicine. Tissues are typically broken down using proteolytic digestion and various mechanical treatments, but success has been limited due to long processing times, low yield, and high manual labor burden. Here, we present a novel microfluidic device that utilizes precision fluid flows to improve the speed and efficiency of tissue digestion. The microfluidic channels were designed to apply hydrodynamic shear forces at discrete locations on tissue specimens up to 1 cm in length and 1 mm in diameter, thereby accelerating digestion through hydrodynamic shear forces and improved enzyme-tissue contact. We show using animal organs that our digestion device with hydro-mincing capabilities was superior to conventional scalpel mincing and digestion based on recovery of DNA and viable single cells. Thus, our microfluidic digestion device can eliminate or reduce the need to mince tissue samples with a scalpel, while reducing sample processing time and preserving cell viability. Another advantage is that downstream microfluidic operations could be integrated to enable advanced cell processing and analysis capabilities. We envision our novel device being used in research and clinical settings to promote single cell-based analysis technologies, as well as to isolate primary, progenitor, and stem cells for use in the fields of tissue engineering and regenerative medicine.

Natural genetic promoters are regulated by multiple cis and trans regulatory factors. For quantitative studies of these promoters, the concentration of only a single factor is typically varied to obtain the dose response or transfer function of the promoters with respect to the factor. Such design of experiments has limited our ability to understand quantitative, combinatorial interactions between multiple regulatory factors at promoters. This limitation is primarily due to the intractable number of experimental combinations that arise from multifactorial design of experiments. To overcome this major limitation, we integrate impact printing and cell-free systems to enable multi-dimensional studies of genetic promoters. We first present a gradient printing system which comprises parallel piezoelectric cantilever beams as a scalable actuator array to generate droplets with tunable volumes in the range of 100 pL-10 nL, which facilitates highly accurate direct dilutions in the range of 1-10 000-fold in a 1 μL drop. Next, we apply this technology to study interactions between three regulatory factors at a synthetic genetic promoter. Finally, a mathematical model of gene regulatory modules is established using the multi-parametric and multi-dimensional data. Our work creates a new frontier in the use of cell-free systems and droplet printing for multi-dimensional studies of synthetic genetic constructs.

Nucleic acids encode the information of life, programming cellular functions and dictating many biological outcomes. Differentiating between cells based on their nucleic acid programs is, thus, a powerful way to unravel the genetic bases of many phenotypes. This is especially important considering that most cells exist in heterogeneous populations, requiring them to be isolated before they can be studied. Existing flow cytometry techniques, however, are unable to reliably recover specific cells based on nucleic acid content. Nucleic acid cytometry is a new field built on droplet microfluidics that allows robust identification, sorting, and sequencing of cells based on specific nucleic acid biomarkers. This review highlights applications that immediately benefit from the approach, biological questions that can be addressed for the first time with it, and considerations for building successful workflows.

Nucleic acids encode the information of life, programming cellular functions and dictating many biological outcomes. Differentiating between cells based on their nucleic acid programs is, thus, a powerful way to unravel the genetic bases of many phenotypes. This is especially important considering that most cells exist in heterogeneous populations, requiring them to be isolated before they can be studied. Existing flow cytometry techniques, however, are unable to reliably recover specific cells based on nucleic acid content. Nucleic acid cytometry is a new field built on droplet microfluidics that allows robust identification, sorting, and sequencing of cells based on specific nucleic acid biomarkers. This review highlights applications that immediately benefit from the approach, biological questions that can be addressed for the first time with it, and considerations for building successful workflows.

Short-lived radiolabeled tracers for positron emission tomography (PET) must be rapidly synthesized, purified, and formulated into injectable solution just prior to imaging. Current radiosynthesizers are generally designed for clinical use, and the HPLC purification and SPE formulation processes often result in a final volume that is too large for preclinical and emerging in vitro applications. Conventional technologies and techniques for reducing this volume tend to be slow, resulting in radioactive decay of the product, and often require manual handling of the radioactive materials. We present a fully-automated microfluidic system based on sweeping gas membrane distillation to rapidly perform the concentration and formulation process. After detailed characterization of the system, we demonstrate fast and efficient concentration and formulation of several PET tracers, evaluate residual solvent content to establish the safety of the formulated tracers for injection, and show that the formulated tracer can be used for in vivo imaging.

Organ-on-a-chip systems possess a promising future as drug screening assays and as testbeds for disease modeling in the context of both single-organ systems and multi-organ-chips. Although it comprises approximately one fourth of the body weight of a healthy human, an organ frequently overlooked in this context is white adipose tissue (WAT). WAT-on-a-chip systems are required to create safety profiles of a large number of drugs due to their interactions with adipose tissue and other organs via paracrine signals, fatty acid release, and drug levels through sequestration. We report a WAT-on-a-chip system with a footprint of less than 1 mm2 consisting of a separate media channel and WAT chamber connected via small micropores. Analogous to the in vivo blood circulation, convective transport is thereby confined to the vasculature-like structures and the tissues protected from shear stresses. Numerical and analytical modeling revealed that the flow rates in the WAT chambers are less than 1/100 of the input flow rate. Using optimized injection parameters, we were able to inject pre-adipocytes, which subsequently formed adipose tissue featuring fully functional lipid metabolism. The physiologically relevant microfluidic environment of the WAT-chip supported long term culture of the functional adipose tissue for more than two weeks. Due to its physiological, highly controlled, and computationally predictable character, the system has the potential to be a powerful tool for the study of adipose tissue associated diseases such as obesity and type 2 diabetes.

Isolation and enumeration of circulating tumor cells (CTCs) from blood is important for determining patient prognosis and monitoring treatment. Methods based on affinity to cell surface markers have been applied to both purify (via immunoseparation) and identify (via immunofluorescence) CTCs. However, variability of cell biomarker expression associated with tumor heterogeneity and evolution and cross-reactivity of antibody probes have long complicated CTC enrichment and immunostaining. Here, we report a truly label-free high-throughput microfluidic approach to isolate, enumerate, and characterize the biophysical properties of CTCs using an integrated microfluidic device. Vortex-mediated deformability cytometry (VDC) consists of an initial vortex region which enriches large CTCs, followed by release into a downstream hydrodynamic stretching region which deforms the cells. Visualization and quantification of cell deformation with a high-speed camera revealed populations of large (>15 μm diameter) and deformable (aspect ratio >1.2) CTCs from 16 stage IV lung cancer samples, that are clearly distinguished by increased deformability compared to contaminating blood cells and rare large cells isolated from healthy patients. The VDC technology demonstrated a comparable positive detection rate of putative CTCs above healthy baseline (93.8%) with respect to standard immunofluorescence (71.4%). Automation allows full enumeration of CTCs from a 10 mL vial of blood within

New tools for applying force to animals, tissues, and cells are critically needed in order to advance the field of mechanobiology, as few existing tools enable simultaneous imaging of tissue and cell deformation as well as cellular activity in live animals. Here, we introduce a novel microfluidic device that enables high-resolution optical imaging of cellular deformations and activity while applying precise mechanical stimuli to the surface of the worms cuticle with a pneumatic pressure reservoir. To evaluate device performance, we compared analytical and numerical simulations conducted during the design process to empirical measurements made with fabricated devices. Leveraging the well-characterized touch receptor neurons (TRNs) with an optogenetic calcium indicator as a model mechanoreceptor neuron, we established that individual neurons can be stimulated and that the device can effectively deliver steps as well as more complex stimulus patterns. This microfluidic device is therefore a valuable platform for investigating the mechanobiology of living animals and their mechanosensitive neurons.

Cytology of sparingly available cell samples from both clinical and experimental settings would benefit from high-selectivity protein tools. To minimize cell handling losses in sparse samples, we design a multi-stage assay using a lab-on-a-disc that integrates cell handling and subsequent single-cell western blotting (scWestern). As the two-layer microfluidic device rotates, the induced centrifugal force directs dissociated cells to dams, which in turn localize the cells over microwells. Cells then sediment into the microwells, where the cells are lysed and subjected to scWestern. Taking into account cell losses from loading, centrifugation, and lysis-buffer exchange, our lab-on-a-disc device handles cell samples with as few as 200 cells with 75% cell settling efficiencies. Over 70% of microwells contain single cells after the centrifugation. In addition to cell settling efficiency, cell-size filtration from a mixed population of two cell lines is also realized by tuning the cell time-of-flight during centrifugation (58.4% settling efficiency with 6.4% impurity). Following the upstream cell handling, scWestern analysis detects four proteins (GFP, β-TUB, GAPDH, and STAT3) in a glioblastoma cell line. By integrating the lab-on-a-disc cell preparation and scWestern analysis, our platform measures proteins from sparse cell samples at single-cell resolution.

Water transport across epithelial monolayers is of central importance in mammalian fluid homeostasis, and epithelial aquaporin (AQP) water channels are potential drug targets. Current methods to measure transepithelial water permeability based on indicator dilution have limited accuracy and can require hours for a single measurement. We report here a microfluidics platform for rapid and accurate measurement of water transport across a conventionally cultured epithelial monolayer on a porous filter requiring only a single image obtained using a standard laboratory fluorescence microscope. The undersurface of a porous polyester filter containing cultured epithelial cells on top is contacted with a perfused microfluidic channel of 100 μm width, 20 μm height and 10 cm length with folded geometry, with in-plane size of 3.2 × 3.2 mm2 for visualization with a 2× objective lens. Osmotic water permeability is measured from the steady-state concentration profile along the length of the channel of a membrane-impermeant fluorescent dye in the perfusate, in which an osmotic gradient is imposed by an anisosmolar solution overlying the epithelial monolayer; diffusional water permeability is measured using a D2O/H2O-sensing fluorescent dye in the perfusate with a D2O-containing isosmolar solution overlying the cell layer. Permeability values are deduced from single fluorescence images. The method, named fluid transport on a chip (FT-on-Chip), was applied to measure transepithelial osmotic and diffusional water permeability in control and AQP4-expressing epithelial cell monolayers. FT-on-Chip allows for rapid, accurate and repeated measurements of transepithelial water permeability, and is generalizable to transport measurements of ions and solutes using suitable indicator dyes.

There is a growing awareness that complex 3-dimensional (3D) organs are not well represented by monolayers of a single cell type - the standard format for many drug screens. To address this deficiency, and with the goal of improving screens so that drugs with good efficacy and low toxicity can be identified, microphysiological systems (MPS) are being developed that better capture the complexity of in vivo physiology. We have previously described an organ-on-a-chip platform that incorporates perfused microvessels, such that survival of the surrounding tissue is entirely dependent on delivery of nutrients through the vessels. Here we describe an arrayed version of the platform that incorporates multiple vascularized micro-organs (VMOs) on a 96-well plate. Each VMO is independently-addressable and flow through the micro-organ is driven by hydrostatic pressure. The platform is easy to use, requires no external pumps or valves, and is highly reproducible. As a proof-of-concept we have created arrayed vascularized micro tumors (VMTs) and used these in a blinded screen to assay a small library of compounds, including FDA-approved anti-cancer drugs, and successfully identified both anti-angiogenic and anti-tumor drugs. This 3D platform is suitable for efficacy/toxicity screening against multiple tissues in a more physiological environment than previously possible.

Characterization of blood flow rheology in hematological disorders is critical for understanding disease pathophysiology. Existing methods tomeasure blood rheological parameters are limited in their physiological relevance, and there is a need for new tools that focus on the microcirculation and extract properties at finer resolution than overall flow resistance. Herein, we present a method that combines microfluidic systems and powerful object-tracking computational technologies with mathematical modeling to separate the red blood cell flow profile into a bulk component and a wall component. We use this framework to evaluate differential contributions of effective viscosity and wall friction to the overall resistance in blood from patients with sickle cell disease (SCD) under a range of oxygen tensions. Our results demonstrate that blood from patients with SCD exhibits elevated frictional and viscous resistances at all physiologic oxygen tensions. Additionally, the viscous resistance increases more rapidly than the frictional resistance as oxygen tension decreases, which may confound analyses that extract only flow velocities or overall flow resistances. Furthermore, we evaluate the impact of transfusion treatments on the components of the resistance, revealing patient variability in blood properties that may improve our understanding of the heterogeneity of clinical responses to such treatments. Overall, our systemprovides a newmethod to analyze patient-specific blood properties and can be applied to awide range of hematological and vascular disorders. [ABSTRACT FROM AUTHOR]

Coculturing multiple cell types together in 3-dimensional (3D) cultures better mimics the in vivo microphysiological environment, and has become widely adopted in recent years with the development of organ-on-chip systems. However, a bottleneck in set-up of these devices arises as a result of the delivery of the gel into the microfluidic chip being sensitive to pressure fluctuations, making gel confinement at a specific region challenging, especially when manual operation is performed. In this paper, we present a novel design of an on-chip regulator module with pressure-releasing safety microvalves that can facilitate stable gel delivery into designated microchannel regions while maintaining well-controlled, non-bursting gel interfaces. This pressure regulator design can be integrated into different microfluidic chip designs and is compatible with a wide variety of gel injection apparatuses operated automatically or manually at different flow rates. The sensitivity and working range of this pressure regulator can be adjusted by changing the width of its pressure releasing safety microvalve design. The effectiveness of the design is validated by its incorporation into a microfluidic platform we have developed for generating 3D vascularized micro-organs (VMOs). Reproducible gel loading is demonstrated for both an automatic syringe pump and a manually-operated micropipettor. This design allows for rapid and reproducible loading of hydrogels into microfluidic devices without the risk of bursting gel-air interfaces.

This paper reports a method for generating an intact and perfusable microvascular network that connects to microfluidic channels without appreciable leakage. This platform incorporates different stages of vascular development including vasculogenesis, endothelial cell (EC) lining, sprouting angiogenesis, and anastomosis in sequential order. After formation of a capillary network inside the tissue chamber via vasculogenesis, the adjacent microfluidic channels are lined with a monolayer of ECs, which then serve as the high-pressure input ("artery") and low pressure output ("vein") conduits. To promote a tight interconnection between the artery/vein and the capillary network, sprouting angiogenesis is induced, which promotes anastomosis of the vasculature inside the tissue chamber with the EC lining along the microfluidic channels. Flow of fluorescent microparticles confirms the perfusability of the lumenized microvascular network, and minimal leakage of 70 kDa FITC-dextran confirms physiologic tightness of the EC junctions and completeness of the interconnections between artery/vein and the capillary network. This versatile device design and its robust construction methodology establish a physiological transport model of interconnected perfused vessels from artery to vascularized tissue to vein. The system has utility in a wide range of organ-on-a-chip applications as it enables the physiological vascular interconnection of multiple on-chip tissue constructs that can serve as disease models for drug screening.

Multiphysics microfluidics, which combines multiple functional physical processes in a microfluidics platform, is an emerging research area that has attracted increasing interest for diverse biomedical applications. Multiphysics microfluidics is expected to overcome the limitations of individual physical phenomena through combining their advantages. Furthermore, multiphysics microfluidics is superior for cell manipulation due to its high precision, better sensitivity, real-time tunability, and multi-target sorting capabilities. These exciting features motivate us to review this state-of-the-art field and reassess the feasibility of coupling multiple physical processes. To confine the scope of this paper, we mainly focus on five common forces in microfluidics: inertial lift, elastic, dielectrophoresis (DEP), magnetophoresis (MP), and acoustic forces. This review first explains the working mechanisms of single physical phenomena. Next, we classify multiphysics techniques in terms of cascaded connections and physical coupling, and we elaborate on combinations of designs and working mechanisms in systems reported in the literature to date. Finally, we discuss the possibility of combining multiple physical processes and associated design schemes and propose several promising future directions. [ABSTRACT FROM AUTHOR]

In this work, we report for the first time a simple and robust method for constructing a 3D microfluidic analytical device on a single piece of hydrophobic cotton cloth. Specifically, laser scanning technology was applied to process hydrophilic regions at the top and bottom of a single piece of hydrophobic cloth. Symmetrical hydrophilic regions at the bottom and top constituted vertical microfluidic channels, and asymmetrical hydrophilic regions constituted transverse flow channels. Liquid flow velocity in 3D cloth-based microchannels can be adjusted flexibly by modifying laser parameters, and programmable laser scanning can be utilized to process 3D microfluidic devices with various patterns. Single-piece 3D cloth-based microfluidic devices formed via this method can be used in many fields such as information encryption and anti-counterfeiting, multi-liquid printing and liquid mixing dilution. Compared to traditional processing methods of 3D cloth-based microfluidic devices, the laser scanning method eliminates multiple complex and repetitive assembly processes, which is a significant advance in this research area. This processing method provides a new option for fast and large-scale manufacturing of 3D cloth-based microfluidic analysis devices for point-of-care testing application in undeveloped regions/countries. [ABSTRACT FROM AUTHOR]

There remains an unmet need for a simple microfluidic platform that can perform multi-step and multi-reagent biochemical assays in parallel for high-throughput detection and analysis of single molecules and single cells. In response, we report herein a PDMS-based vacuum-driven microfluidic array that is capable of multi-step sample loading and digitalization. The array features multi-level bifurcation microchannels connecting to 4096 dead-end microchambers for partitioning liquid reagents/samples. To realize multi-step repetitive liquid sample loading, we attach an external vacuum onto the chip to create internal negative pressure for a continuous liquid driving force. We demonstrated a high uniformity of our device for three sequential liquid loadings. To further improve its utility, we developed a thermosetting-oil covering method to prevent evaporation for assays that require high temperatures. We successfully performed digital PCR assays on our device, demonstrating the efficient multi-step reagent handling and the effective anti-evaporation design for thermal cycling. Furthermore, we performed a digital PCR detection for single-cell methicillin-resistant Staphylococcus aureus using a three-step loading approach and achieved accurate single-cell quantification. Taken together, we have demonstrated that our vacuum-driven microfluidic array is capable of multi-step sample digitalization at high throughput for single-molecule and single-cell analyses. [ABSTRACT FROM AUTHOR]

With the prevalence of bacterial infections and increasing levels of antibiotic resistance comes the need for rapid and accurate methods for bacterial classification (BC) and antibiotic susceptibility testing (AST). Here we demonstrate the use of the fluid handling technique digital microfluidics (DMF) for automated and simultaneous BC and AST using growth metabolic markers. Custom instrumentation was developed for this application including an integrated heating module and a machine-learning-enabled low-cost colour camera for real-time absorbance and fluorescent sample monitoring on multipurpose devices. Antibiotic dilutions along with sample handling, mixing and incubation at 37 °C were all pre-programmed and processed automatically. By monitoring the metabolism of resazurin, resorufin beta- D -glucuronide and resorufin beta- D -galactopyranoside to resorufin, BC and AST were achieved in under 18 h. AST was validated in two uropathogenic E. coli strains with antibiotics ciprofloxacin and nitrofurantoin. BC was performed independently and simultaneously with ciprofloxacin AST for E. coli, K. pneumoniae, P. mirabilis and S. aureus. Finally, a proof-of-concept multiplexed system for breakpoint testing of two antibiotics, as well as E. coli and coliform classification was investigated with a multidrug-resistant E. coli strain. All bacteria were correctly identified, while AST and breakpoint test results were in essential and category agreement with reference methods. These results show the versatility and accuracy of this all-in-one microfluidic system for analysis of bacterial growth and phenotype. [ABSTRACT FROM AUTHOR]

Translation of advanced cell-based assays exhibiting a higher degree of automation, miniaturization, and integration of complementary sensing functions is mainly limited by the development of industrial-relevant prototypes that can be readily produced in larger volumes. Despite the increasing number of academic publications in recent years, the manufacturability of these microfluidic cell cultures systems is largely ignored, thus severely restricting their implementation in routine toxicological applications. We have developed a dual-sensor integrated microfluidic cell analysis platform using industrial specifications, materials, and fabrication methods to conduct risk assessment studies of engineered nanoparticles to overcome this academic–industrial gap. Non-invasive and time-resolved monitoring of cellular oxygen uptake and metabolic activity (pH) in the absence and presence of nanoparticle exposure is accomplished by integrating optical sensor spots into a cyclic olefin copolymer (COC)-based microfluidic platform. Results of our nanotoxicological study, including two physiological cell barriers that are essential in the protection from exogenous factors, the intestine (Caco-2) and the vasculature (HUVECs) showed that the assessment of the cells' total energy metabolism is ideally suited to rapidly detect cytotoxicities. Additional viability assay verification using state-of-the-art dye exclusion assays for nanotoxicology demonstrated the similarity and comparability of our results, thus highlighting the benefits of employing a compact and cost-efficient microfluidic dual-sensor platform as a pre-screening tool in nanomaterial risk assessment and as a rapid quality control measure in medium to high-throughput settings. [ABSTRACT FROM AUTHOR]

Cell membrane water permeability is an important determinant of epithelial fluid secretion, tissue swelling, angiogenesis, tumor spread and other biological processes. Cellular water channels, aquaporins, are important drug targets. Water permeability is generally measured from the kinetics of cell volume change in response to an osmotic gradient. Here, we developed a microfluidic platform in which cells expressing a cytoplasmic, volume-sensing fluorescent dye are rapidly subjected to an osmotic gradient by solution mixing inside a ~0.1 nL droplet surrounded by oil. The solution mixing time was