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Rapid tests to assess the susceptibility of bacteria to antibiotics are required to inform antibiotic stewardship. We have developed a novel test, which measures changes in the impedance of a 100 nanoliter volume of bacterial suspension to determine an “electrical” minimum inhibitory concentration (eMIC). Two representative strains of Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus were tested against a panel of frontline antibiotics with different modes of action (ciprofloxacin, doxycycline, colistin and imipenem, gentamicin, and ceftazidime). The eMIC measured at 1 h correlated strongly with a standard 24 h microbroth dilution MIC for all combinations of antibiotics and bacteria, allowing strains to be correctly assigned as sensitive or resistant measured in a fraction of the time.

A droplet generator has been developed that interfaces with a barrier-on-chip platform for temporal analyte compartmentalisation and analysis. Droplets are generated every 20 minutes in 8 separate parallel microchannels, with an average droplet volume of 9.47 ± 0.6 μL, allowing simultaneous analysis of 8 different experiments. The device was tested using an epithelial barrier model by monitoring the diffusion of a fluorescent high molecular weight dextran molecule. The epithelial barrier was perturbed using detergent leading to a peak at 3–4 hours, correlating with simulations. For the untreated (control) a constant, very low level of dextran diffusion was observed. The epithelial cell barrier properties were also continuously measured using electrical impedance spectroscopy to extract an equivalent trans epithelial resistance.

We describe fractionation of sub-micron vesicles and particles suspended in high conductivity electrolytes using an electrokinetically biased Deterministic Lateral Displacement (DLD) device. An optimised, asymmetric array of micron-sized pillars and gaps, with an AC electric field applied orthogonal to the fluid flow gives an approximately ten-fold reduction in the intrinsic critical diameter (Dc) of the device. The asymmetry in the device maximises the throughput. Fractionation of populations of 100 nm and 400 nm extruded vesicles is achieved in 690 mS m−1 KCl, and 100 nm, 200 nm and 500 nm polystyrene particles in 105 mS m−1 KCl. The electrokinetically biased DLD may provide solutions for simple and rapid isolation of extracellular vesicles

We experimentally study the repulsion of charged microscopic particles with the channel walls during electrophoresis in microfluidic devices. For low frequencies of the electric fields (< 10 kHz), this repulsion is mainly due to the hydrodynamic interaction caused by the flow vortices that arise from the slip velocity induced by the electric field on the particle surface, as shown in a recent publication [Fernandez-Mateo et al., Physical Review Letters, 128, 074501, (2022)]. The maximum slip velocity on the particle surface is inferred from measurements of wall-particle separation. Importantly, this procedure allows us to infer very small slip velocities that otherwise are too weak to be measured directly. Data at small electric field amplitudes (E0) agree with theoretical predictions using the model of Concentration Polarization Electroosmosis (CPEO), which has recently been proposed as the mechanism behind the flow vortices on the surface of the particles. Data for higher electric fields show that the predictions of the CPEO theory for weak electric fields are not valid beyond E0 ∼ 60 kV/m. Additionally, we also show that, for sufficiently strong electric fields, the quadrupolar flow structures become disrupted, leading to a weaker wall repulsion. Ministerio de Ciencia e Innovación PGC2018-099217-B-I00, 10.13039/501100011033

We describe a user-friendly, open source software for single-particle detection/counting in a continuous-flow. The tool automatically processes video images of particles, including pre-conditioning, followed by size-based discrimination for independent detection of fluorescent and non-fluorescent particles of different sizes. This is done by interactive tuning of a reduced set of parameters that can be checked with a robust, real-time quality control of the original video files. The software provides a concentration distribution of the particles in the transverse direction of the fluid flow. The software is a versatile tool for many microfluidic applications and does not require expertise in image analysis.

Breast milk is essential for facilitating the growth and development of infants and for providing immune protection against viral infections in the infant’s airways. Yet, regulation of inflammation by milk components may be needed to reduce immune pathology. While milk-derived extracellular vesicles (EVs) are bestowed with immunomodulatory capacities, their role in bronchial epithelial barrier function and inflammation has not yet been examined. We hypothesised that during feeding, milk is not only ingested, but aerosols containing milk EVs are inhaled and locally delivered to the infant’s airways to suppress aberrant inflammation. A bronchial epithelial model of viral infection was used to explore the direct effect of milk EVs on cellular barrier function and cytokine release during stimulation with a viral dsRNA analogue (Poly I:C). We demonstrate that milk EVs improved the dsRNA-mediated decrease in ionic barrier integrity, limited tight junction reorganisation and reduced inflammatory cytokine production (IL-6, IL-8 and TNF-α). This protective response was EV-mediated, could be successfully titrated and exhibited a time-dependent response. The results indicate that if EV-containing milk aerosols are inhaled during feeding, this may lead to protection of the airway integrity from adverse inflammatory effects.

A multichannel microfluidic platform for real-time monitoring of epithelial barrier integrity by electrical impedance has been developed. Growth and polarization of human epithelial cells from the airway or gastrointestinal tract was continuously monitored over 5 days in 8 parallel, individually perfused microfluidic chips. Electrical impedance data were continuously recorded to monitor cell barrier formation using a low-cost bespoke impedance analyser. Data was analysed using an electric circuit model to extract the equivalent transepithelial electrical resistance and epithelial cell layer capacitance. The cell barrier integrity steadily increased overtime, achieving an average resistance of 418 ± 121 Ω cm 2 (airway cells) or 207 ± 59 Ω cm 2 (gastrointestinal cells) by day 5. The utility of the polarized airway epithelial barrier was demonstrated using a 24 hour challenge with double stranded RNA to mimic viral infection. This caused a rapid decrease in barrier integrity in association with disruption of tight junctions, whereas simultaneous treatment with a corticosteroid reduced this effect. The platform is able to measure barrier integrity in real-time and is scalable, thus has the potential to be used for drug development and testing.

Microfluidic impedance cytometry is a label-free technique for high-throughput single-cell analysis. Multi-frequency impedance measurements provide data that allows full characterisation of cells, linking electrical phenotype to individual biophysical properties. To efficiently extract the information embedded in the electrical signals, potentially in real-time, tailored signal processing is needed. Artificial intelligence approaches provide a promising new direction. Here we demonstrate the ability of neural networks to decipher impedance cytometry signals in two challenging scenarios: (i) to determine the intrinsic dielectric properties of single cells directly from raw impedance data streams, (ii) to capture single-cell signals that are hidden in the measured signals of coincident cells. The accuracy of the results and the high processing speed (fractions of ms per cell) demonstrate that neural networks can have an important role in impedance-based single-cell analysis.

Electrophoresis describes the motion of charged particles suspended in electrolytes when subjected to an external electric field. Previous experiments have shown that particles undergoing electrophoresis are repelled from nearby channel walls, contrary to the standard description of electrophoresis that predicts no hydrodynamic repulsion. Dielectrophoretic (DEP) repulsive forces have been commonly invoked as the cause of this wall repulsion. We show that DEP forces can only account for this wall repulsion at high frequencies of applied electric field. In the presence of a low-frequency field, quadrupolar electro-osmotic flows are observed around the particles. We experimentally demonstrate that these hydrodynamic flows are the cause of the widely observed particle-wall interaction. This hydrodynamic wall repulsion should be considered in the design and application of electric-field-driven manipulation of particles in microfluidic devices. Ministerio de Ciencia e Innovación PGC2018-099217-B-I00, 10.13039/501100011033

There is an urgent need to develop simple and fast antimicrobial susceptibility tests (ASTs) that allow informed prescribing of antibiotics. Here, we describe a label-free AST that can deliver results within an hour, using an actively dividing culture as starting material. The bacteria are incubated in the presence of an antibiotic for 30 min, and then approximately 105 cells are analysed one-by-one with microfluidic impedance cytometry for 2–3 min. The measured electrical characteristics reflect the phenotypic response of the bacteria to the mode of action of a particular antibiotic, in a 30-minute incubation window. The results are consistent with those obtained by classical broth microdilution assays for a range of antibiotics and bacterial species., There is an urgent need to develop simple and fast antimicrobial susceptibility tests. Here, Spencer et al. describe a label-free test that can deliver results within an hour, consisting of a 30-min antibiotic treatment followed by single-cell analysis of phenotypic responses with microfluidic impedance cytometry.

This dataset supports the publication in Electrophoresis entitled 'Short Communication: A simple and accurate method of measuring the Zeta-Potential of microfluidic channels'. This is the original dataset used to create Figures 2B, 2C and Figure 3. It also contains the uncertainty estimations of the measured zeta potential displayed in Figure 3 computed from the original dataset for each of the conductivity solutions used in the work: 1.5 mS/m, 5.2 mS/m and 11.4 mS/m.

Objectives: The provision of high-quality personal protective equipment (PPE) has been a critical challenge during the COVID-19 pandemic. We evaluated an alternative strategy, mass deployment of a powered air-purifying respirator (PeRSo), in a large university hospital.Methods: We performed prospective user feedback via questionnaires sent to healthcare workers (HCWs) issued PeRSos, economic analysis, and evaluated the real-world impact.Results: Where paired responses were available, PeRSo was preferred over droplet precautions for comfort, patient response, overall experience, and subjective feeling of safety. For all responses, more participants reported the overall experience being rated “Very good” more frequently for PeRSo. The primary limitation identified was impairment of hearing. Economic simulation exercises revealed that the adoption of PeRSo within ICU is associated with net cost savings in the majority of scenarios and savings increased progressively with greater ITU occupancy. In evaluation during the second UK wave, over 3,600 respirators were deployed, all requested by staff, which were associated with a low staff absence relative to most comparator hospitals.Conclusions: Health services should consider a widespread implementation of powered reusable respirators as a safe and sustainable solution for the protection of HCWs as SARS-CoV-2 becomes an endemic viral illness.

Organ on a chip or microphysiological systems (MPSs) aim to resolve current challenges surrounding drug discovery and development resulting from an unrepresentative static cell culture or animal models that are traditionally used by generating a more physiologically relevant environment. Many different airway MPSs have been developed that mimic alveolar or bronchial interfaces, but few methods for aerosol drug delivery at the air–liquid interface exist. This work demonstrates a compact Surface Acoustic Wave (SAW) drug delivery device that generates an aerosol of respirable size for delivery of compounds directly onto polarized or differentiated epithelial cell cultures within an airway barrier MPS and conventional static inserts. As proof of principle, the SAW drug delivery device was used to nebulize viral dsRNA analog poly I:C and steroids fluticasone and dexamethasone without disrupting their biological function.

Respiratory diseases account for over 5 million deaths yearly and are a huge burden to healthcare systems worldwide. Murine models have been of paramount importance to decode human lung biologyin vivo, but their genetic, anatomical, physiological and immunological differences with humans significantly hamper successful translation of research into clinical practice. Thus, to clearly understand human lung physiology, development, homeostasis and mechanistic dysregulation that may lead to disease, it is essential to develop models that accurately recreate the extraordinary complexity of the human pulmonary architecture and biology. Recent advances in micro-engineering technology and tissue engineering have allowed the development of more sophisticated models intending to bridge the gap between the native lung and its replicatesin vitro. Alongside advanced culture techniques, remarkable technological growth in downstream analyses has significantly increased the predictive power of human biology-basedin vitromodels by allowing capture and quantification of complex signals. Refined integrated multi-omics readouts could lead to an acceleration of the translational pipeline fromin vitroexperimental settings to drug development and clinical testing in the future. This review highlights the range and complexity of state-of-the-art lung models for different areas of the respiratory system, from nasal to large airways, small airways and alveoli, with consideration of various aspects of disease states and their potential applications, including pre-clinical drug testing. We explore how development of optimised physiologically relevantin vitrohuman lung models could accelerate the identification of novel therapeutics with increased potential to translate successfully from the bench to the patient's bedside.

Electric fields are commonly used for manipulating particles and liquids in microfluidic systems. In this work, we report stationary electro-osmotic flow vortices around dielectric micropillars induced by ac electric fields in electrolytes. The flow characteristics are theoretically predicted based on the well-known phenomena of surface conductance and concentration polarization around a charged object. The stationary flows arise from two distinct contributions working together: an oscillating nonuniform zeta potential induced around the pillar and a rectified electric field induced by the ion concentration gradients. We refer to this fluid flow as concentration-polarization electro-osmosis (CPEO). We present experimental data in support of the theoretical predictions. The magnitude and frequency dependence of the electro-osmotic velocity are in agreement with the theoretical estimates and are significantly different from predictions based on the standard theory for induced-charge electro-osmosis, which has previously been postulated as the origin of the stationary flow around dielectric objects. In addition to furthering our understanding of the influence of ac fields on fluid flows, we anticipate that this work will also expand the use of ac fields for flow control in microfluidic systems. European Regional Development Fund (ERDF) and the Spanish Research Agency MCI PGC2018-099217-B-I00

We experimentally demonstrate quadrupolar electro-osmotic flows around charged dielectric microspheres immersed in an electrolyte when subjected to an alternating current electric field. We present an electrokinetic model that predicts the flow characteristics based on the phenomena of surface conductance and polarization of the electrolyte concentration around the particles. We refer to these flows as concentration polarization electro-osmosis. We anticipate that these flows may play a major role in the electric-field-induced assembly of colloids and on the electrokinetic manipulation of dielectric micro- and nanoparticles.

Electric fields are commonly used to trap and separate micro- and nanoparticles near channel constrictions in microfluidic devices. The trapping mechanism is attributed to the electrical forces arising from the nonhomogeneous electric field caused by the constrictions, and the phenomenon is known as insulator-based-dielectrophoresis (iDEP). In this paper, we describe stationary electroosmotic flows of electrolytes around insulating constrictions induced by low frequency AC electric fields (below 10 kHz). Experimental characterization of the flows is described for two different channel heights (50 and 10 μm), together with numerical simulations based on an electrokinetic model that considers the modification of the local ionic concentration due to surface conductance on charged insulating walls. We term this phenomenon concentration-polarization electroosmosis (CPEO). The observed flow characteristics are in qualitative agreement with the predictions of this model. However, for shallow channels (10 μm), trapping of the particles on both sides of the constrictions is also observed. This particle and fluid behavior could play a major role in iDEP and could be easily misinterpreted as a dielectrophoretic force.

ObjectivesThe study aims to determine the relative importance of key attributes of a novel intrauterine device. The device monitors uterine oxygen, pH and temperature in real time with the aim of improving our understanding and treatment of reproductive disorders.DesignA discrete choice experiment was used to elicit preferences in this novel investigative tool. The attributes and levels used in the choice scenarios were length of time using the device (7, 14 or 28 days), information obtained to guide treatment (limited, majority or all cases), risk of complications (1% or 10%) and discreteness (completely discrete, moderately discrete or indiscrete).SettingSecondary care hospital in Hampshire, UK.Participants361 women of reproductive age.Primary and secondary outcome measuresConditional logit and latent class logit regression models to determine the preference for each attribute.ResultsConditional logit coefficients allow comparison between attributes; women placed most importance on obtaining information to guide treatment in all cases (2.771), followed by having a completely discrete device (1.104), reducing risk of complications by 1% (0.184) and decreased length of time by 1 day (0.0150). All coefficients pConclusionsWomen placed most importance on having a device that obtains information to guide treatment and are willing to use the device for a longer, have a device with higher risk of complications and an indiscrete device if it is able to provide answers and direction for treatment of their reproductive disorder.

The differences in behaviour between individual cells in a large population are often important, yet are masked in bulk analyses where only average parameters are measured. One unresolved question in the field of immunology is the extent to which important immunological phenomena such as immunodominance to cancer antigens correlates with the average activity of a population of antigen-specific T lymphocytes, or with the activity of individual "outlier" cells. Despite progress in single cell technologies, few platforms are available that can deliver time-resolved, functional analysis at single cell resolution, for these investigations. We have developed an accessible high-throughput platform to measure single T cell signalling in real time following time-controlled stimulation by live antigen presenting cells. The cell-trap array consists of thousands of individual microwells cast in an agarose block, which is biocompatible and permeable to nutrients. Single T cells are isolated in wells via passive sedimentation and size exclusion, achieving up to 90% occupancy. The device enables simultaneous activation of thousands of single CD8+ cells. Stimulation with soluble reagents (ionomycin, anti-CD3 antibodies) or antigen presenting cells leads to changes in intracellular calcium concentrations which were measured using calcium-chelating fluorophore dyes. The platform was used to demonstrate a range of activation profiles among individual cells of a cloned, antigen specific CD8+ T cell hybridoma in response to both nonspecific stimuli and specific, physiologically relevant antigen stimulation. The presence of two different activation profiles was demonstrated, together with rare outlier behaviour among cells that are essentially clonal.

The Publisher regrets that this article is an accidental duplication of an article that has already been published, http://dx.doi.org/10.1016/j.jinf.2020.04.025. The duplicate article has therefore been withdrawn. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/our-business/policies/article-withdrawal.

The SARS-CoV-2 virus infection is a rapidly spreading global pandemic. Recent media coverage has highlighted the importance of protecting health-care workers together with issues surrounding availability and suitability of Personal Protective Equipment (PPE). Around 20% of healthcare workers treating COVID19 cases in Italy have become infected which leads to staff absence at a critical point during the pandemic, and unfortunately in some cases mortality.PPE plays a major role in control programs. Standard PPE such as N95/FFP3 facemasks have limitations such as an ineffective seal during talking or after prolonged use, face shapes which cannot be adequately fitted, and logistical issues ensuring availability of the correct mask for each person. Furthermore, global stock is low, and issues around diagnostic testing specificity and turnaround time may lead to infectious patients receiving care from health care staff who are not wearing appropriate PPE. To address acute shortcomings in PPE availability, we have developed a simple pressurised air purified respirator unit, incorporating a combination of inexpensive and widely available components parts. The prototype was developed to minimise the number and complexity of manufacturing steps with the intention that derivative versions could be developed in many different parts of the world, including low resource settings with minor modification, where transmission could be rapid amongst high population densities.The “Personal Respirator – Southampton” (PeRSo) delivers HEPA filtered air from a battery powered fan-filter assembly through a lightweight hood/face mask that can be comfortably worn for several hours. Initial user feedback provided by doctors and nurses shows the PeRSo prototype was preferred to standard N95/FFP3 masks, being more comfortable, reducing time lost placing and removing PPE between patients, and allowing better communication. Preliminary tests indicate that the device removes microbes and passes the “fit tests” widely used to evaluate face masks. Full verification of the safety and the duration of effectiveness and durability of the device is required, as part of translation into use. Rapid upscale of production is required to protect healthcare workers from infection while the global situation accelerates, so that they can look after patients during the peak of the pandemic.

Single-cell impedance cytometry is a label-free analysis technique that is now widely used to measure the electrical properties of a cell and to differentiate different subpopulations. Current techniques are limited to measuring the impedance of a single cell at one or two simultaneous frequencies. Also, there are no methods that extrapolate the intrinsic electrical properties of single cells. We demonstrate a new approach that uses multifrequency impedance measurements to determine the complete intrinsic electrical properties of thousands of single cells at high throughput. The applicability of the method is demonstrated by measuring the properties of red blood cells and red cell ghosts, deriving the unique values of conductivity and permittivity of the membrane and cytoplasm for each individual cell.

Deterministic Lateral Displacement (DLD) is a microfluidic technique where arrays of micropillars within a microchannel deflect particles leading to size-based segregation. We recently demonstrated that applying AC electric fields orthogonal to the fluid flow increases the separation capabilities of these devices with a deflection angle that depends on the electric field magnitude and frequency. Particle deviation occurs in two distinct regimes depending on frequency. At high frequencies particles deviate due to negative dielectrophoresis (DEP). At low frequencies (below 1 kHz) particles oscillate perpendicular to the flow direction due to electrophoresis and are also deflected within the device. Significantly, the threshold electric field magnitude for the low frequency deviation is much lower than for deflection at high frequencies by DEP. In order to characterize the enhanced separation at low frequencies, the induced deviation was compared between the two frequency ranges. For high frequencies, we develop both theoretically and experimentally scaling laws for the dependence of particle deviation on several parameters, namely the amplitude of the applied voltage, particle size and liquid velocity where DEP forces compete with viscous drag. A novel theoretical framework is presented that enables simulation of particle trajectories subjected to DEP forces in DLD devices. Deviation angles predicted by simulations are in very good agreement with experimental data. At low frequencies (below 1 kHz), particles follow the same scaling law, but with much lower voltages. This indicates that electrokinetic phenomena other than DEP play an important role in driving particle behaviour. Experiments show that at low frequencies, particle motion is affected by quadrupolar electrohydrodynamic flows around the insulating pillars of the DLD array. We quantify the difference between the two frequency regimes and show that an electrokinetic model based only on DEP forces is limited to frequencies of 1 kHz and above. Agencia Estatal de Investigación PGC2018-099217-B-I00