On the compatibility of single-cell microcarriers (nanovials) with microfluidic impedance cytometry.

We investigate for the first time the compatibility of nanovials with microfluidic impedance cytometry (MIC). Nanovials are suspendable crescent-shaped single-cell microcarriers that enable specific cell adhesion, the creation of compartments for undisturbed cell growth and secretion, as well as protection against wall shear stress. MIC is a label-free single-cell technique that characterizes flowing cells based on their electrical fingerprints and it is especially targeted to cells that are naturally in suspension. Combining nanovial technology with MIC is intriguing as it would represent a robust framework for the electrical analysis of single adherent cells at high throughput. Here, as a proof-of-concept, we report the MIC analysis of mesenchymal stromal cells loaded in nanovials. The electrical analysis is supported by numerical simulations and validated by means of optical analysis. We demonstrate that the electrical diameter can discriminate among free cells, empty nanovials, cell-loaded nanovials, and clusters, thus grounding the foundation for the use of nanovials in MIC. Furthermore, we investigate the potentiality of MIC to assess the electrical phenotype of cells loaded in nanovials and we draw directions for future studies.

Multichannel impedance cytometry downstream of cell separation by deterministic lateral displacement to quantify macrophage enrichment in heterogeneous samples.

The integration of on-chip biophysical cytometry downstream of microfluidic enrichment for inline monitoring of phenotypic and separation metrics at single-cell sensitivity can allow for active control of separation and its application to versatile sample sets. We present integration of impedance cytometry downstream of cell separation by deterministic lateral displacement (DLD) for enrichment of activated macrophages from a heterogeneous sample, without the problems of biased sample loss and sample dilution caused by off-chip analysis. This required designs to match cell/particle flow rates from DLD separation into the confined single-cell impedance cytometry stage, the balancing of flow resistances across the separation array width to maintain unidirectionality, and the utilization of co-flowing beads as calibrated internal standards for inline assessment of DLD separation and for impedance data normalization. Using a heterogeneous sample with un-activated and activated macrophages, wherein macrophage polarization during activation causes cell size enlargement, on-chip impedance cytometry is used to validate DLD enrichment of the activated subpopulation at the displaced outlet, based on the multiparametric characteristics of cell size distribution and impedance phase metrics. This hybrid platform can monitor separation of specific subpopulations from cellular samples with wide size distributions, for active operational control and enhanced sample versatility.

Rapid Assessment of Susceptibility of Bacteria and Erythrocytes to Antimicrobial Peptides by Single-Cell Impedance Cytometry.

Antimicrobial peptides (AMPs) represent a promising class of compounds to fight antibiotic-resistant infections. In most cases, they kill bacteria by making their membrane permeable and therefore exhibit low propensity to induce bacterial resistance. In addition, they are often selective, killing bacteria at concentrations lower than those at which they are toxic to the host. However, clinical applications of AMPs are hindered by a limited understanding of their interactions with bacteria and human cells. Standard susceptibility testing methods are based on the analysis of the growth of a bacterial population and therefore require several hours. Moreover, different assays are required to assess the toxicity to host cells. In this work, we propose the use of microfluidic impedance cytometry to explore the action of AMPs on both bacteria and host cells in a rapid manner and with single-cell resolution. Impedance measurements are particularly well-suited to detect the effects of AMPs on bacteria, due to the fact that the mechanism of action involves perturbation of the permeability of cell membranes. We show that the electrical signatures of Bacillus megaterium cells and human red blood cells (RBCs) reflect the action of a representative antimicrobial peptide, DNS-PMAP23. In particular, the impedance phase at high frequency (e.g., 11 or 20 MHz) is a reliable label-free metric for monitoring DNS-PMAP23 bactericidal activity and toxicity to RBCs. The impedance-based characterization is validated by comparison with standard antibacterial activity assays and absorbance-based hemolytic activity assays. Furthermore, we demonstrate the applicability of the technique to a mixed sample of B. megaterium cells and RBCs, which paves the way to study AMP selectivity for bacterial versus eukaryotic cells in the presence of both cell types.

Extensional-Flow Impedance Cytometer for Contactless and Optics-Free Erythrocyte Deformability Analysis.

OBJECTIVE: Deformability is an essential feature of red blood cells (RBCs), enabling them to undergo significant shape change in response to external forces. Impaired erythrocyte deformability is associated with several pathologic conditions, and quantitative measurement of RBC deformability is critical to understanding and diagnosing RBC related diseases. Whereas traditional approaches to cell mechanical characterization generally have limited throughput, emerging microscale technologies are opening new opportunities for high-throughput deformability cytometry at the single-cell level. METHODS: In this work, we propose an innovative microfluidic system based on (i) a hyperbolic microchannel to induce erythrocyte deformation by extensional flow, and (ii) an electrical sensing zone with coplanar electrodes to evaluate the deformed cell shape. RESULTS: RBC deformation under extensional flow is achieved, and the deformed cell shape is quantified by means of an electrical anisotropy index, at a throughput of 300 cell/s. Measurements of healthy and chemically stiffened RBCs demonstrate that the anisotropy index can be used to characterize RBC deformability, as an alternative to deformation indices based on high-speed image processing. CONCLUSION: A contactless and optics-free approach for RBC deformability analysis has been presented. SIGNIFICANCE: Due to its simplicity and potential for integration, the proposed approach holds promises for fast and low-cost erythrocyte deformability assays, especially in point-of-care and resource-limited settings.

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.

Modified Red Blood Cells as Multimodal Standards for Benchmarking Single-Cell Cytometry and Separation Based on Electrical Physiology.

Biophysical cellular information at single-cell sensitivity is becoming increasingly important within analytical and separation platforms that associate the cell phenotype with markers of disease, infection, and immunity. Frequency-modulated electrically driven microfluidic measurement and separation systems offer the ability to sensitively identify single cells based on biophysical information, such as their size and shape, as well as their subcellular membrane morphology and cytoplasmic organization. However, there is a lack of reliable and reproducible model particles with well-tuned subcellular electrical phenotypes that can be used as standards to benchmark the electrical physiology of unknown cell types or to benchmark dielectrophoretic separation metrics of novel device strategies. Herein, the application of red blood cells (RBCs) as multimodal standard particles with systematically modulated subcellular electrophysiology and associated fluorescence level is presented. Using glutaraldehyde fixation to vary membrane capacitance and by membrane resealing after electrolyte penetration to vary interior cytoplasmic conductivity and fluorescence in a correlated manner, each modified RBC type can be identified at single-cell sensitivity based on phenomenological impedance metrics and fitted to dielectric models to compute biophysical information. In this manner, single-cell impedance data from unknown RBC types can be mapped versus these model RBC types for facile determination of subcellular biophysical information and their dielectrophoretic separation conditions, without the need for time-consuming algorithms that often require unknown fitting parameters. Such internal standards for biophysical cytometry can advance in-line phenotypic recognition strategies.

Electro-Optical Classification of Pollen Grains via Microfluidics and Machine Learning.

OBJECTIVE: In aerobiological monitoring and agriculture there is a pressing need for accurate, label-free and automated analysis of pollen grains, in order to reduce the cost, workload and possible errors associated to traditional approaches. METHODS: We propose a new multimodal approach that combines electrical sensing and optical imaging to classify pollen grains flowing in a microfluidic chip at a throughput of 150 grains per second. Electrical signals and synchronized optical images are processed by two independent machine learning-based classifiers, whose predictions are then combined to provide the final classification outcome. RESULTS: The applicability of the method is demonstrated in a proof-of-concept classification experiment involving eight pollen classes from different taxa. The average balanced accuracy is 78.7% for the electrical classifier, 76.7% for the optical classifier and 84.2% for the multimodal classifier. The accuracy is 82.8% for the electrical classifier, 84.1% for the optical classifier and 88.3% for the multimodal classifier. CONCLUSION: The multimodal approach provides better classification results with respect to the analysis based on electrical or optical features alone. SIGNIFICANCE: The proposed methodology paves the way for automated multimodal palynology. Moreover, it can be extended to other fields, such as diagnostics and cell therapy, where it could be used for label-free identification of cell populations in heterogeneous samples.

Single-cell microfluidic impedance cytometry: from raw signals to cell phenotypes using data analytics.

The biophysical analysis of single-cells by microfluidic impedance cytometry is emerging as a label-free and high-throughput means to stratify the heterogeneity of cellular systems based on their electrophysiology. Emerging applications range from fundamental life-science and drug assessment research to point-of-care diagnostics and precision medicine. Recently, novel chip designs and data analytic strategies are laying the foundation for multiparametric cell characterization and subpopulation distinction, which are essential to understand biological function, follow disease progression and monitor cell behaviour in microsystems. In this tutorial review, we present a comparative survey of the approaches to elucidate cellular and subcellular features from impedance cytometry data, covering the related subjects of device design, data analytics (i.e., signal processing, dielectric modelling, population clustering), and phenotyping applications. We give special emphasis to the exciting recent developments of the technique (timeframe 2017-2020) and provide our perspective on future challenges and directions. Its synergistic application with microfluidic separation, sensor science and machine learning can form an essential toolkit for label-free quantification and isolation of subpopulations to stratify heterogeneous biosystems.

A Bayesian Approach for Coincidence Resolution in Microfluidic Impedance Cytometry.

OBJECTIVE: Cell counting and characterization is fundamental for medicine, science and technology. Coulter-type microfluidic devices are effective and automated systems for cell/particle analysis, based on the electrical sensing zone principle. However, their throughput and accuracy are limited by coincidences (i.e., two or more particles passing through the sensing zone nearly simultaneously), which reduce the observed number of particles and may lead to errors in the measured particle properties. In this work, a novel approach for coincidence resolution in microfluidic impedance cytometry is proposed. METHODS: The approach relies on: (i) a microchannel comprising two electrical sensing zones and (ii) a model of the signals generated by coinciding particles. Maximum a posteriori probability (MAP) estimation is used to identify the model parameters and therefore characterize individual particle properties. RESULTS: Quantitative performance assessment on synthetic data streams shows a counting sensitivity of 97% and a positive predictive value of 99% at concentrations of 2×106 particles/ml. An application to red blood cell analysis shows accurate particle characterization up to a throughput of about 2500 particles/s. An original formula providing the expected number of coinciding particles is derived, and good agreement is found between experimental results and theoretical predictions. CONCLUSION: The proposed cytometer enables the decomposition of signals generated by coinciding particles into individual particle contributions, by using a Bayesian approach. SIGNIFICANCE: This system can be profitably used in applications where accurate counting and characterization of cell/particle suspensions over a broad range of concentrations is required.

High-throughput label-free characterization of viable, necrotic and apoptotic human lymphoma cells in a coplanar-electrode microfluidic impedance chip.

The study and the characterization of cell death mechanisms are fundamental in cell biology research. Traditional death/viability assays usually involve laborious sample preparation and expensive equipment or reagents. In this work, we use electrical impedance spectroscopy as a label-free methodology to characterize viable, necrotic and apoptotic human lymphoma U937 cells. A simple three-electrode coplanar layout is used in a differential measurement scheme and thousands of cells are measured at high-throughput (≈200 cell/s). Tailored signal processing enables accurate and robust cell characterization without the need for cell focusing systems. The results suggest that, at low frequency (0.5 MHz), signal magnitude enables the discrimination between viable/necrotic cells and cell fragments, whereas phase information allows discriminating between viable cells and necrotic cells. At higher frequency (10 MHz) two subpopulations of cell fragments are distinguished. This work substantiates the prominent role of electrical impedance spectroscopy for the development of next-generation cell viability assays.

High-throughput electrical position detection of single flowing particles/cells with non-spherical shape.

We present an innovative impedance cytometer for the measurement of the cross-sectional position of single particles or cells flowing in a microchannel. As predicted by numerical simulations and experimentally validated, the proposed approach is applicable to particles/cells with either spherical or non-spherical shape. In particular, the optics-free high-throughput position detection of individual flowing red blood cells (RBCs) is demonstrated and applied to monitor RBCs hydrodynamic focusing under different sheath flow conditions. Moreover, the device provides multiparametric information useful for lab-on-a-chip applications, including particle inter-arrival times and velocity profile, as well as RBCs mean corpuscular volume, distribution width and electrical opacity.

Oil-in-Water fL Droplets by Interfacial Spontaneous Fragmentation and Their Electrical Characterization.

Inkjet printing is here employed for the first time as a method to produce femtoliter-scale oil droplets dispersed in water. In particular, picoliter-scale fluorinated oil (FC40) droplets are printed in the presence of perfluoro-1-octanol surfactant at a velocity higher than 5 m/s. Femtoliter-scale oil droplets in water are spontaneously formed through a fragmentation process at the water/air interface using minute amounts of nonionic surfactant (down to 0.003% v/v of Tween 80). This fragmentation occurs by a Plateau-Rayleigh mechanism at a moderately high Weber number (101). A microfluidic chip with integrated microelectrodes allows droplets characterization in terms of number and diameter distribution (peaked at about 3 µm) by means of electrical impedance measurements. These results show an unprecedented possibility to scale oil droplets down to the femtoliter scale, which opens up several perspectives for a tailored oil-in-water emulsion fabrication for drug encapsulation, pharmaceutic preparations, and cellular biology.

A simple electrical approach to monitor dielectrophoretic focusing of particles flowing in a microchannel.

This paper reports an impedance-based system for the quantitative assessment of dielectrophoretic (DEP) focusing of single particles flowing in a microchannel. Particle lateral positions are detected in two electrical sensing zones placed before and after a DEP-focusing region, respectively. In each sensing zone, particle lateral positions are estimated using the unbalance between the opposite pulses of a differential current signal obtained with a straightforward coplanar electrode configuration. The system is used to monitor the focusing of polystyrene beads of 7 or 10 µm diameter, under various conditions of DEP field intensities and flow rates that produce different degrees of focusing. This electrical approach represents a simple and valuable alternative to optical methods for monitoring of particle focusing systems.

Simulation and performance analysis of a novel high-accuracy sheathless microfluidic impedance cytometer with coplanar electrode layout.

The performance of a novel microfluidic impedance cytometer (MIC) with coplanar configuration is investigated in silico. The main feature of the device is the ability to provide accurate particle-sizing despite the well-known measurement sensitivity to particle trajectory. The working principle of the device is presented and validated by means of an original virtual laboratory providing close-to-experimental synthetic data streams. It is shown that a metric correlating with particle trajectory can be extracted from the signal traces and used to compensate the trajectory-induced error in the estimated particle size, thus reaching high-accuracy. An analysis of relevant parameters of the experimental setup is also presented.

Coplanar electrode microfluidic chip enabling accurate sheathless impedance cytometry.

Microfluidic impedance cytometry offers a simple non-invasive method for single-cell analysis. Coplanar electrode chips are especially attractive due to ease of fabrication, yielding miniaturized, reproducible, and ultimately low-cost devices. However, their accuracy is challenged by the dependence of the measured signal on particle trajectory within the interrogation volume, that manifests itself as an error in the estimated particle size, unless any kind of focusing system is used. In this paper, we present an original five-electrode coplanar chip enabling accurate particle sizing without the need for focusing. The chip layout is designed to provide a peculiar signal shape from which a new metric correlating with particle trajectory can be extracted. This metric is exploited to correct the estimated size of polystyrene beads of 5.2, 6 and 7 µm nominal diameter, reaching coefficient of variations lower than the manufacturers' quoted values. The potential impact of the proposed device in the field of life sciences is demonstrated with an application to Saccharomyces cerevisiae yeast.

Numerical Investigation of a Novel Wiring Scheme Enabling Simple and Accurate Impedance Cytometry.

Microfluidic impedance cytometry is a label-free approach for high-throughput analysis of particles and cells. It is based on the characterization of the dielectric properties of single particles as they flow through a microchannel with integrated electrodes. However, the measured signal depends not only on the intrinsic particle properties, but also on the particle trajectory through the measuring region, thus challenging the resolution and accuracy of the technique. In this work we show via simulation that this issue can be overcome without resorting to particle focusing, by means of a straightforward modification of the wiring scheme for the most typical and widely used microfluidic impedance chip.

High accuracy particle analysis using sheathless microfluidic impedance cytometry.

This paper describes a new design of microfluidic impedance cytometer enabling accurate characterization of particles without the need for focusing. The approach uses multiple pairs of electrodes to measure the transit time of particles through the device in two simultaneous different current measurements, a transverse (top to bottom) current and an oblique current. This gives a new metric that can be used to estimate the vertical position of the particle trajectory through the microchannel. This parameter effectively compensates for the non-uniform electric field in the channel that is an unavoidable consequence of the use of planar parallel facing electrodes. The new technique is explained and validated using numerical modelling. Impedance data for 5, 6 and 7 µm particles are collected and compared with simulations. The method gives excellent coefficient of variation in (electrical) radius of particles of 1% for a sheathless configuration.

A Simple and Robust Event-Detection Algorithm for Single-Cell Impedance Cytometry.

Microfluidic impedance cytometry is emerging as a powerful label-free technique for the characterization of single biological cells. In order to increase the sensitivity and the specificity of the technique, suited digital signal processing methods are required to extract meaningful information from measured impedance data. In this study, a simple and robust event-detection algorithm for impedance cytometry is presented. Since a differential measuring scheme is generally adopted, the signal recorded when a cell passes through the sensing region of the device exhibits a typical odd-symmetric pattern. This feature is exploited twice by the proposed algorithm: first, a preliminary segmentation, based on the correlation of the data stream with the simplest odd-symmetric template, is performed; then, the quality of detected events is established by evaluating their E2O index, that is, a measure of the ratio between their even and odd parts. A thorough performance analysis is reported, showing the robustness of the algorithm with respect to parameter choice and noise level. In terms of sensitivity and positive predictive value, an overall performance of 94.9% and 98.5%, respectively, was achieved on two datasets relevant to microfluidic chips with very different characteristics, considering three noise levels. The present algorithm can foster the role of impedance cytometry in single-cell analysis, which is the new frontier in "Omics."