Viable Escherichia coli enumeration on a polydimethylsiloxane (PDMS) chip with vertical channel-well configuration.

The culture-based methods for viable Escherichia coli (E. coli) detection suffer from long detection time and laborious procedures, whereas the molecule tests and immune recognition technologies lack live/dead E. coli differentiation. Rapid, easy-to-use, and accessible viable E. coli detection is of benefit to bacterial infection diagnosis and risk warning of E. coli contamination of water and food, safeguarding human health. Herein, we propose a microwell chip-based solution to realize simple and rapid determination of viable E. coli. The vertical channel-well configuration is applied to develop the microwell array chip for increasing the microwell density (6200 wells/cm2), yielding a broad dynamic range from 103 to 107 CFU/mL. We incorporate an inducible enzyme assay with the developed chip and achieve the differentiation of live/dead E. coli within 4 h, significantly shortening the detection time from over 24 h in the standard method. By encapsulating single E. coli into microwells, the concentration of viable cells can be determined simultaneously through counting positive microwells. In addition, the air soluble PDMS that can store negative pressure for independent sample digitalization endows the developed chip with simple operation and less reliance on external equipment. With further developments for increasing the number of microwell and integrating more sample panels, the developed chip can become a useful tool for rapid viable E. coli enumeration with user-friendly operation, simple procedures, and accessibility in decentralized settings, thereby deploying this device for water and food safety monitoring, as well as clinical bacterial infection diagnosis.

Inoculum size-insensitive susceptibility determination of urine sample based on in-situ measurement of inducible enzyme activity after 20 min of antibiotic exposure.

BACKGROUND: The empirical antibiotic therapies for bacterial infections cause the emergence and propagation of multi-drug resistant bacteria, which not only impair the effectiveness of existing antibiotics but also raise healthcare costs. To reduce the empirical treatments, rapid antimicrobial susceptibility testing (AST) of causative microorganisms in clinical samples should be conducted for prescribing evidence-based antibiotics. However, most of culture-based ASTs suffer from inoculum effect and lack differentiation of target pathogen and commensals, hampering their adoption for evidence-based antibiotic prescription. Therefore, rapid ASTs which can specifically determine pathogens' susceptibilities, regardless of the bacterial load in clinical samples, are in urgent need. RESULTS: We present a pathogen-specific and inoculum size-insensitive AST to achieve the reliable susceptibility determination on Escherichia coli (E. coli) in urine samples. The developed AST is featured with an 1 h sample-to-result workflow in a filter, termed on-filter AST. The AST results can be obtained by using an inducible enzymatic assay to in-situ measure the cell response of E. coli collected from urine after 20 min of antibiotic exposure. The calculated detection limit of our AST (1.95 × 104 CFU/mL) is much lower than the diagnosis threshold of urinary tract infections. The specific expression of the inducible enzyme enables on-filter AST to correctly profile the susceptibilities of target pathogen to multi-type antibiotics without the interference from commensals. We performed the on-filter AST on 1 mL urine samples with bacterial loads varying from 105 CFU/mL to 107 CFU/mL and compared the results to that of standard method, demonstrating its insensitivity to inoculum size. SIGNIFICANCE: The developed AST is demonstrated to be of high sensitivity, specificity, and insensitive to inoculum size. With further developments for additional bacteria and clinical validation, on-filter AST is promising as a rapid and reliable surrogate of culture-based AST to promote the evidence-based prescription at the first visit and minimize the emergency of new multi-drug resistant microorganisms.

Enzymatic Antimicrobial Susceptibility Testing with Bacteria Identification in 30 min.

Rapid antimicrobial susceptibility testing (AST) with the ability of bacterial identification is urgently needed for evidence-based antibiotic prescription. Herein, we propose an enzymatic AST (enzyAST) that employs ß-d-glucuronidase as a biomarker to identify pathogens and profile phenotypic susceptibilities simultaneously. EnzyAST enables to offer binary AST results within 30 min, much faster than standard methods (>16 h). The general applicability of enzyAST was verified by testing the susceptibility of two Escherichia coli strains to three antibiotics with different action mechanisms. The pilot study also shows that the minimal inhibitory concentrations can be determined by enzyAST with the statistical analysis of enzymatic activity of the bacteria population exposed to varying antibiotic concentrations. With further development of multiple bacteria and sample treatment, enzyAST could be able to evaluate the susceptibility of pathogens in clinical samples directly to facilitate the evidence-based therapy.

An On-Chip Viscoelasticity Sensor for Biological Fluids.

There are so many non-Newtonian fluids in our daily life, such as milk, blood, cytoplasm, and mucus, most of which are viscoelastic heterogeneous liquid containing cells, inorganic ion, metabolites, and hormones. In microfluidic microparticle-manipulating applications, the target particles are practically distributed within the biological fluids like blood and urine. The viscoelasticity of biological fluid is constantly ignored for simplicity especially when the fluid is substantially diluted and contains rather complex components. However, even the fluid's ultraweak viscoelasticity actually affects the microparticle migration and may bring a completely different behavior compared with the Newtonian fluids. As a result, a robust and easy operated on-chip viscoelasticity sensor is potential and desired in many research and industrial fields, including assay sample preparation, clinical diagnostics, and on-chip sensor. In this work, we employed stable non-Newtonian fluid-polyethylene oxide (PEO) solutions with various concentrations to investigate and calibrate effects of the weak fluidic viscoelasticity on microparticle behaviors in a double-layered microfluidic channel. An analogy-based database of fluidic patterns for viscoelasticity sensing and relaxation time measurement was established. Then, we tested different biological fluids including blood plasma and fetal bovine serum and proved that they exhibited similar viscoelasticity effects to the PEO solutions with the corresponding concentration, which reached a good agreement with available results by references. The detection limitation of relaxation time can reach 1 ms. It promised a robust and integrated on-chip microfluidic viscoelasticity sensor for different biological fluids without complicated calculations.

All-In-One Escherichia coli Viability Assay for Multi-dimensional Detection of Uncomplicated Urinary Tract Infections.

The urinary tract infections by antibiotic-resistant bacteria have been a serious public health problem and increase the healthcare costs. The conventional technologies of diagnosis and antimicrobial susceptibility testing (AST) relying on multiple culture-based assays are time-consuming and labor-intensive and thus compel the empirical antimicrobial therapies to be prescribed, fueling the prevalence of antimicrobial resistance. Herein, we propose an all-in-one Escherichia coli viability assay in an enclosed 3D microwell array chip, termed digital ß-d-glucuronidase (GUS)-AST assay. It employs GUS, a specific metabolism-related enzyme, to convert the presence of E. coli into bright fluorescence. The random distribution of single bacteria in microwell array enables to quantify the E. coli concentrations by counting the positive microwells. We incorporate the most probable number with digital quantification to lower the limit of detection and expand the dynamic range to 7 orders. The digital GUS-AST assay is able to indicate the potency of antibiotics and determine the minimum inhibitory concentrations. A streamlined procedure of urine removal, bacterial separation, and digital GUS-AST is established to perform the direct analysis of bacteria population in urine. The sample-to-result workflow can be finished in 4.5 h with a limit of detection of 39 CFU/mL. With further development for additional pathogens and multiple antibiotic conditions, the digital GUS-AST assay could help physicians to prescribe timely targeted therapies for better patient outcomes and the minimum emergence of resistant bacteria.

Mechanism and Effects of Cellular Creep in a Microfluidic Filter.

Biomicroparticles such as proteins, bacterium, and cells are known to be viscoelastic, which significantly affects their performance in microfluidic applications. However, the exact effects and the quantitative study of cellular viscoelastic creep within different applications remain unclear. In this study, the cellular-deforming evolution within a filter unit was studied using a multiphysics numerical model. A general cellular creep deformation process of viscoelastic particle trapping in pores was revealed. Two featured variables, namely, the maximum surface displacement and the volumetric strain, were identified and determined to quantitatively describe the evolution. The effects of flow conditions and physical characteristics of the microparticles were studied. Furthermore, a Giardia concentration experiment was conducted using an integrated hydraulic filtration system with a porous membrane. The experimental results agreed well with the numerical analysis, indicating that, compared to pure elastic particles, it is more difficult to release cellular material matters including cells, chemical synthetic particles, and microbes from trapping due to their time-accumulated creep deformation.

Sheathless Separation of Cyanobacterial Anabaena by Shape Using Viscoelastic Microfluidics.

Cyanobacteria have a wide range of impact on natural ecosystems, and have been recognized as potentially rich sources of pharmacological and structurally interesting secondary metabolites. To better understand the basic molecular processes and mechanisms that influence and regulate the growth (like length) of cyanobacteria, or connections between environment, genotype, and phenotype, it would be essential to separate shape-synchronized cyanobacterial cell populations with relatively uniform length and size. This work proposes a novel and efficient method to separate cyanobacterial Anabaena by shape (rod aspect ratio) using viscoelastic microfluidics in a straight channel with expansion-contraction cavity arrays (ECCA channel). The biocompatible viscoelastic solutions with dissolved polymer would induce a combined effect of inertial lift force, elastic force, and secondary drag force for Anabaena flowing in it. Therefore, Anabaena with different lengths reach different lateral equilibrium positions and flow out from different outlets. Factors including flow rate, fluid viscoelasticity, channel structure, and length on the shape-based cell separation were studied systematically. This work, for the first time, demonstrates continuous and sheathless shape-based separation of cyanobacteria using viscoelastic microfluidics. Moreover, its ability to manipulate objects with different morphologies and with a size of >100 µm will extend the capability of microfluidics to a completely new field that has never been reached and would be attractive across a range of new applications.

Mechanical Strain-Enabled Reconstitution of Dynamic Environment in Organ-on-a-Chip Platforms: A Review.

Organ-on-a-chip (OOC) uses the microfluidic 3D cell culture principle to reproduce organ- or tissue-level functionality at a small scale instead of replicating the entire human organ. This provides an alternative to animal models for drug development and environmental toxicology screening. In addition to the biomimetic 3D microarchitecture and cell-cell interactions, it has been demonstrated that mechanical stimuli such as shear stress and mechanical strain significantly influence cell behavior and their response to pharmaceuticals. Microfluidics is capable of precisely manipulating the fluid of a microenvironment within a 3D cell culture platform. As a result, many OOC prototypes leverage microfluidic technology to reproduce the mechanically dynamic microenvironment on-chip and achieve enhanced in vitro functional organ models. Unlike shear stress that can be readily generated and precisely controlled using commercial pumping systems, dynamic systems for generating proper levels of mechanical strains are more complicated, and often require miniaturization and specialized designs. As such, this review proposes to summarize innovative microfluidic OOC platforms utilizing mechanical actuators that induce deflection of cultured cells/tissues for replicating the dynamic microenvironment of human organs.

Modular and Self-Contained Microfluidic Analytical Platforms Enabled by Magnetorheological Elastomer Microactuators.

Portability and low-cost analytic ability are desirable for point-of-care (POC) diagnostics; however, current POC testing platforms often require time-consuming multiple microfabrication steps and rely on bulky and costly equipment. This hinders the capability of microfluidics to prove its power outside of laboratories and narrows the range of applications. This paper details a self-contained microfluidic device, which does not require any external connection or tubing to deliver insert-and-use image-based analysis. Without any microfabrication, magnetorheological elastomer (MRE) microactuators including pumps, mixers and valves are integrated into one modular microfluidic chip based on novel manipulation principles. By inserting the chip into the driving and controlling platform, the system demonstrates sample preparation and sequential pumping processes. Furthermore, due to the straightforward fabrication process, chips can be rapidly reconfigured at a low cost, which validates the robustness and versatility of an MRE-enabled microfluidic platform as an option for developing an integrated lab-on-a-chip system.

On-Chip Optical Detection of Viruses: A Review.

The current outbreak of the coronavirus disease-19 (COVID-19) pandemic worldwide has caused millions of fatalities and imposed a severe impact on our daily lives. Thus, the global healthcare system urgently calls for rapid, affordable, and reliable detection toolkits. Although the gold-standard nucleic acid amplification tests have been widely accepted and utilized, they are time-consuming and labor-intensive, which exceedingly hinder the mass detection in low-income populations, especially in developing countries. Recently, due to the blooming development of photonics, various optical chips have been developed to detect single viruses with the advantages of fast, label-free, affordable, and point of care deployment. Herein, optical approaches especially in three perspectives, e.g., flow-free optical methods, optofluidics, and surface-modification-assisted approaches, are summarized. The future development of on-chip optical-detection methods in the wave of emerging new ideas in nanophotonics is also briefly discussed.

Correction: Modular off-chip emulsion generator enabled by a revolving needle.

Correction for 'Modular off-chip emulsion generator enabled by a revolving needle' by Yuxin Zhang et al., Lab Chip, 2020, 20, 4592-4599, DOI: 10.1039/D0LC00939C.

Modular off-chip emulsion generator enabled by a revolving needle.

Microfluidic chips have demonstrated unparalleled abilities in droplet generation, including precise control over droplet size and monodispersity. And yet, their rather complicated microfabrication process and operation can be a barrier for inexperienced researchers, which hinders microdroplets from unleashing their potential in broader fields of research. Here, we attempt to remove this barrier by developing an integrated and modular revolving needle emulsion generator (RNEG) to achieve high-throughput production of uniformly sized droplets in an off-chip manner. The RNEG works by driving a revolving needle to pinch the dispersed phase in a minicentrifuge tube. The system is constructed using modular components without involving any microfabrication, thereby enabling user-friendly operation. The RNEG is capable of producing microdroplets of various liquids with diameters ranging from tens to hundreds of micrometres. We further examine the principle of operation using numerical simulations and establish a simple model to predict the droplet size. Moreover, by integrating curing and centrifugation processes, the RNEG can produce hydrogel microparticles and transfer them from an oil phase into a water phase. Using this ability, we demonstrate the encapsulation and culture of single yeast cells within hydrogel microparticles. We envisage that the RNEG can become a versatile and powerful tool for high-throughput production of emulsions to facilitate diverse biological and chemical research.

A Review of Secondary Flow in Inertial Microfluidics.

Inertial microfluidic technology, which can manipulate the target particle entirely relying on the microchannel characteristic geometry and intrinsic hydrodynamic effect, has attracted great attention due to its fascinating advantages of high throughput, simplicity, high resolution and low cost. As a passive microfluidic technology, inertial microfluidics can precisely focus, separate, mix or trap target particles in a continuous and high-flow-speed manner without any extra external force field. Therefore, it is promising and has great potential for a wide range of industrial, biomedical and clinical applications. In the regime of inertial microfluidics, particle migration due to inertial effects forms multiple equilibrium positions in straight channels. However, this is not promising for particle detection and separation. Secondary flow, which is a relatively minor flow perpendicular to the primary flow, may reduce the number of equilibrium positions as well as modify the location of particles focusing within channel cross sections by applying an additional hydrodynamic drag. For secondary flow，the pattern and magnitude can be controlled by the well-designed channel structure, such as curvature or disturbance obstacle. The magnitude and form of generated secondary flow are greatly dependent on the disturbing microstructure. Therefore, many inventive and delicate applications of secondary flow in inertial microfluidics have been reported. In this review, we comprehensively summarize the usage of the secondary flow in inertial microfluidics.

Sheathless separation of microalgae from bacteria using a simple straight channel based on viscoelastic microfluidics.

Microalgae cells have been recognized as a promising sustainable resource to meet worldwide growing demands for renewable energy, food, livestock feed, water, cosmetics, pharmaceuticals, and materials. In order to ensure high-efficiency and high-quality production of biomass, biofuel, or bio-based products, purification procedures prior to the storage and cultivation of the microalgae from contaminated bacteria are of great importance. The present work proposed and developed a simple, sheathless, and efficient method to separate microalgae Chlorella from bacteria Bacillus Subtilis in a straight channel using the viscoelasticity of the medium. Microalgae and bacteria migrate to different lateral positions closer to the channel centre and channel walls respectively. Fluorescent microparticles with 1 µm and 5 µm diameters were first used to mimic the behaviours of bacteria and microalgae to optimize the separating conditions. Subsequently, size-based separation in Newtonian fluid and in viscoelastic fluid in straight channels with different aspect ratios was compared and demonstrated. Under the optimal condition, the removal ratio for 1 µm microparticles and separation efficiency for 5 µm particles can reach up to 98.28% and 93.85% respectively. For bacteria and microalgae cells separation, the removal ratio for bacteria and separation efficiency for microalgae cells is 92.69% and 100% respectively. This work demonstrated the continuous and sheathless separation of microalgae from bacteria for the first time by viscoelastic microfluidics. This technique can also be applied as an efficient and user-friendly method to separate mammalian cells or other kinds of cells.

Liquid metal-filled magnetorheological elastomer with positive piezoconductivity.

Conductive elastic composites have been used widely in soft electronics and soft robotics. These composites are typically a mixture of conductive fillers within elastomeric substrates. They can sense strain via changes in resistance resulting from separation of the fillers during elongation. Thus, most elastic composites exhibit a negative piezoconductive effect, i.e. the conductivity decreases under tensile strain. This property is undesirable for stretchable conductors since such composites may become less conductive during deformation. Here, we report a liquid metal-filled magnetorheological elastomer comprising a hybrid of fillers of liquid metal microdroplets and metallic magnetic microparticles. The composite's resistivity reaches a maximum value in the relaxed state and drops drastically under any deformation, indicating that the composite exhibits an unconventional positive piezoconductive effect. We further investigate the magnetic field-responsive thermal properties of the composite and demonstrate several proof-of-concept applications. This composite has prospective applications in sensors, stretchable conductors, and responsive thermal interfaces.

High-Throughput, Off-Chip Microdroplet Generator Enabled by a Spinning Conical Frustum.

Although droplet-based microfluidics has been broadly used as a versatile tool in biology, chemistry, and nanotechnology, its rather complicated microfabrication process and the requirement of specialized hardware and operating skills hinder researchers fully unleashing the potential of this powerful platform. Here, we develop an integrated microdroplet generator enabled by a spinning conical frustum for the versatile production of near-monodisperse microdroplets in a high-throughput and off-chip manner. The construction and operation of this generator are simple and straightforward without the need of microfabrication, and we demonstrate that the generator is able to passively and actively control the size of the produced microdroplets. In addition to water microdroplets, this generator can produce microdroplets of liquid metal that would be difficult to produce in conventional microfluidic platforms as liquid metal has high surface tension. Moreover, we demonstrate that this generator can produce solid hydrogel microparticles and fibers using integrated ultraviolet (UV) light. In the end, we further explore the ability of this generator for forming double emulsions by coflowing two immiscible liquids. Given the remarkable abilities demonstrated by this platform and the tremendous potential of microdroplets, this user-friendly method may revolutionize the future of droplet-based chemical synthesis and biological analysis.

Fundamentals of Differential Particle Inertial Focusing in Symmetric Sinusoidal Microchannels.

Focusing and separation of particles such as cells at high throughput is extremely attractive for biomedical applications. Particle manipulation based on inertial effects requires a high flow speed and thus is well-suited to high-throughput applications. Recently, inertial focusing and separation using curvilinear microchannels has been attracting a great amount of interest because of the linear structure for parallelization, small device footprint, superior particle-focusing performance, and easy implementation of particle separation. However, the curvature directions of these microchannels alternate, leading to variations in both the magnitude and direction of the induced secondary flow. Accumulation of this variation along the channel causes unpredictable behaviors of particles. This paper systematically investigates the inertial-focusing phenomenon in low-aspect-ratio symmetric sinusoidal channels. First, we comprehensively studied the effects of parameters such as viscosity, flow conditions, particle size, and geometric dimensions of the microchannel on differential particle focusing. We found that particle inertial focusing is generally independent of fluid kinematic viscosity but highly dependent on particle size, flow conditions, and channel dimensions. Next, we derived an explicit scaling factor and included all four dimensionless parameters (particle-blockage ratio, curvature ratio, Dean number, and channel aspect ratio) in a single operational map to illustrate the particle-focusing patterns. Finally, we proposed a rational guideline to intuitively instruct the design of channel dimensions for separation of a given particle mixture.

Microfluidic Mass Production of Stabilized and Stealthy Liquid Metal Nanoparticles.

Functional nanoparticles comprised of liquid metals, such as eutectic gallium indium (EGaIn) and Galinstan, present exciting opportunities in the fields of flexible electronics, sensors, catalysts, and drug delivery systems. Methods used currently for producing liquid metal nanoparticles have significant disadvantages as they rely on both bulky and expensive high-power sonication probe systems, and also generally require the use of small molecules bearing thiol groups to stabilize the nanoparticles. Herein, an innovative microfluidics-enabled platform is described as an inexpensive, easily accessible method for the on-chip mass production of EGaIn nanoparticles with tunable size distributions in an aqueous medium. A novel nanoparticle-stabilization approach is reported using brushed polyethylene glycol chains with trithiocarbonate end-groups negating the requirements for thiol additives while imparting a "stealth" surface layer. Furthermore, a surface modification of the nanoparticles is demonstrated using galvanic replacement and conjugation with antibodies. It is envisioned that the demonstrated microfluidic technique can be used as an economic and versatile platform for the rapid production of liquid metal-based nanoparticles for a range of biomedical applications.

Simple, low-cost fabrication of semi-circular channel using the surface tension of solder paste and its application to microfluidic valves.

This work presents a simple, low-cost method to fabricate semi-circular channels using solder paste, which can amalgamate the cooper surface to form a half-cylinder mold using the surface tension of Sn-Pd alloy (the main component in solder paste). This technique enables semi-circular channels to be manufactured with different dimensions. These semi-circular channels will then be integrated with a polymethylmethacrylate frame and machine screws to create miniaturized, portable microfluidic valves for sequential liquid delivery and particle synthesis. This approach avoids complicated fabrication processes and expensive facilities and thus has the potential to be a useful tool for lab-on-a-chip applications.

Liquid metal-based amalgamation-assisted lithography for fabrication of complex channels with diverse structures and configurations.

Numerous lab-on-a-chip applications benefit from channels with complex structures and configurations in the areas of tissue engineering and clinical diagnostics. The current fabrication approaches require time-consuming, complicated processes and bulky, expensive facilities. In this work, we propose a novel method for the fabrication of complex channels with the assistance of amalgamation of liquid metal with copper tape. This new technique enables the rapid fabrication of liquid metal molds with various dimensions and diverse structures. Two proof-of-concept experiments were conducted to verify the utilization of this method. First, the channel replicated from the liquid metal mold is used to enhance the mixing performance of liquids flowing through the channel. Second, a channel with a semicircular cross-section is fabricated to achieve 3D focusing in a simple way. This proposed technique can be readily used for fabricating complex channels for a wide range of applications.