Concentration-polarization electroosmosis for particle fractionation.

Concentration-polarization electroosmosis (CPEO) refers to steady-state electroosmotic flows around charged dielectric micro-particles induced by low-frequency AC electric fields. Recently, these flows were shown to cause repulsion of colloidal particles from the wall of a microfluidic channel when an electric field is applied along the length of the channel. In this work, we exploit this mechanism to demonstrate fractionation of micron-sized polystyrene particles and bacteria in a flow-focusing device. The results are in agreement with predictions of the CPEO theory. The ease of implementation of CPEO-based fractionation in microfluidics makes it an ideal candidate for combining with current techniques commonly used to generate particle lift, such as inertial or viscoelastic focusing, requiring no extra fabrication steps other than inserting two electrodes.

Insulating traveling-wave electrophoresis.

Traveling-wave electrophoresis (TWE) is a method for transporting charged colloidal particles used in many microfluidic techniques for particle manipulation and fractionation. This method exploits the traveling-wave components of the electric field generated by an array of electrodes subjected to ac voltages with a phase delay between neighboring electrodes. In this article, we propose an alternative way of generating traveling-wave electric fields in microchannels. We apply a rotating electric field around a cylindrical insulating micropillar and the resulting traveling-wave modes induce particle drift around the cylinder. We term this phenomenon insulating traveling-wave electrophoresis (i-TWE) to distinguish it from standard TWE performed with arrays of microelectrodes. We characterized the particle drift experimentally and show a quantitative comparison of the particle velocity with theoretical predictions. Excellent agreement is found when the influence of electro-osmosis on the channel walls is also considered.

Low-frequency electrokinetics in a periodic pillar array for particle separation.

Deterministic Lateral Displacement (DLD) exploits periodic arrays of pillars inside microfluidic channels for high-precision sorting of micro- and nano-particles. Previously we demonstrated how DLD separation can be significantly improved by the addition of AC electrokinetic forces, increasing the tunability of the technique and expanding the range of applications. At high frequencies of the electric field (>1 kHz) the behaviour of such systems is dominated by Dielectrophoresis (DEP), whereas at low frequencies the particle behaviour is much richer and more complex. In this article, we present a detailed numerical analysis of the mechanisms governing particle motion in a DLD micropillar array in the presence of a low-frequency AC electric field. We show how a combination of Electrophoresis (EP) and Concentration-Polarisation Electroosmosis (CPEO) driven wall-particle repulsion account for the observed experimental behaviour of particles, and demonstrate how this complete model can predict conditions that lead to electrically induced deviation of particles much smaller than the critical size of the DLD array.

Droplet fluidics for time-dependent analysis of barrier permeability in an epithelial barrier on chip system.

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.

Electrical Broth Micro-Dilution for Rapid Antibiotic Resistance Testing.

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.

Electrokinetic deterministic lateral displacement for fractionation of vesicles and nano-particles.

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.

Integrating an aerosolized drug delivery device with conventional static cultures and a dynamic airway barrier microphysiological system.

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.

Towards an artificial human lung: modelling organ-like complexity to aid mechanistic understanding.

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 biology in 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 replicates in vitro Alongside advanced culture techniques, remarkable technological growth in downstream analyses has significantly increased the predictive power of human biology-based in vitro models by allowing capture and quantification of complex signals. Refined integrated multi-omics readouts could lead to an acceleration of the translational pipeline from in vitro experimental 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 relevant in vitro human lung models could accelerate the identification of novel therapeutics with increased potential to translate successfully from the bench to the patient's bedside.

Organ-on-a-chip: current gaps and future directions.

As an emerging hot topic of the last decade, Organ on Chip (OoC) is a new technology that is attracting interest from both basic and translational scientists. The Biochemical Society, with its mission of supporting the advancement of science, with addressing grand challenges that have societal impact, has included OoC into their agenda to review the current state of the art, bottlenecks and future directions. This conference brought together representatives of the main stakeholders in the OoC field including academics, end-users, regulators and technology developers to discuss and identify requirements for this new technology to deliver on par with the expectations and the key challenges and gaps that still need to be addressed to achieve robust human-relevant tools, able to positively impact decision making in the pharmaceutical industry and reduce overreliance on poorly predictive animal models.

Real-time monitoring of epithelial barrier function by impedance spectroscopy in a microfluidic platform.

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 cm2 (airway cells) or 207 ± 59 Ω cm2 (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.

Discrete choice experiment exploring women's preferences in a novel device designed to monitor the womb environment and improve our understanding of reproductive disorders.

OBJECTIVES: The 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. DESIGN: A 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). SETTING: Secondary care hospital in Hampshire, UK. PARTICIPANTS: 361 women of reproductive age. PRIMARY AND SECONDARY OUTCOME MEASURES: Conditional logit and latent class logit regression models to determine the preference for each attribute. RESULTS: Conditional 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 p<0.01. Latent class conditional logit assigns participants to two classes with 27.4% in class 1 who are less likely to have higher education or qualify for National Health Service-funded in vitro fertilisation compared with class 2. Those in class 2 placed 1.7 times more importance on a device whose information guided treatment in all cases and a 1% decrease in complications risk was nearly 15 times more attractive. CONCLUSIONS: Women 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.

Deciphering impedance cytometry signals with neural networks.

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.

Wall Repulsion of Charged Colloidal Particles during Electrophoresis in Microfluidic Channels.

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.

Short communication: A simple and accurate method of measuring the zeta-potential of microfluidic channels.

We describe an improved method for determining the electroosmotic mobility and zeta potential of surfaces based on a current-monitoring method. This technique eliminates the requirement for measurements of channel dimensions and sample conductivities, leading to a simple high precision measurement. The zeta potential of PDMS is measured for native surfaces and surfaces treated with a nonionic surfactant in low-conductivity electrolytes.

Concentration-Polarization Electroosmosis near Insulating Constrictions within Microfluidic Channels.

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.

A Personal Respirator to Improve Protection for Healthcare Workers Treating COVID-19 (PeRSo).

Introduction: SARS-CoV-2 infection is a global pandemic. Personal Protective Equipment (PPE) to protect healthcare workers has been a recurrent challenge in terms of global stocks, supply logistics and suitability. In some settings, around 20% of healthcare workers treating COVID-19 cases have become infected, which leads to staff absence at peaks of the pandemic, and in some cases mortality. Methods: To address shortcomings in PPE, we developed a simple powered air purifying respirator, made from inexpensive and widely available components. The prototype was designed to minimize manufacturing complexity so that derivative versions could be developed in low resource settings with minor modification. Results: The "Personal Respirator - Southampton" (PeRSo) delivers High-Efficiency Particulate Air (HEPA) filtered air from a battery powered fan-filter assembly into a lightweight hood with a clear visor that can be comfortably worn for several hours. Validation testing demonstrates that the prototype removes microbes, avoids excessive CO2 build-up in normal use, and passes fit test protocols widely used to evaluate standard N95/FFP2 and N99/FFP3 face masks. Feedback from doctors and nurses indicate the PeRSo prototype was preferred to standard FFP2 and FFP3 masks, being more comfortable and reducing the time and risk of recurrently changing PPE. Patients report better communication and reassurance as the entire face is visible. Conclusion: Rapid upscale of production of cheaply produced powered air purifying respirators, designed to achieve regulatory approval in the country of production, could protect healthcare workers from infection and improve healthcare delivery during the COVID-19 pandemic.

Powered Respirators Are Effective, Sustainable, and Cost-Effective Personal Protective Equipment for SARS-CoV-2.

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.

A fast impedance-based antimicrobial susceptibility test.

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.

Time-resolved microwell cell-pairing array reveals multiple T cell activation profiles.

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.

Electrokinetic biased deterministic lateral displacement: scaling analysis and simulations.

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.