Micro-impedance cytometry for detection and analysis of micron-sized particles and bacteria

The sensitivity of a microfluidic impedance flow cytometer is governed by the dimensions of the sample analysis volume. A small volume gives a high sensitivity, but this can lead to practical problems including fabrication and clogging of the device. We describe a microfluidic impedance cytometer which uses an insulating fluid to hydrodynamically focus a sample stream of particles suspended in electrolyte, through a large sensing volume. The detection region consists of two pairs of electrodes fabricated within a channel 200 μm wide and 30 μm high. The focussing technique increases the sensitivity of the system without reducing the dimensions of the microfluidic channel. We demonstrate detection and discrimination of 1 μm and 2 μm diameter polystyrene beads and also Escherichia coli. Impedance data from single particles are correlated with fluorescence emission measured simultaneously. Data are also compared with conventional flow cytometry and dynamic light scattering: the coefficient of variation (CV) of size is found to be comparable between the systems.     

Simultaneous high speed optical and impedance analysis of single particles with a microfluidic cytometer

We describe a microfluidic cytometer that performs simultaneous optical and electrical characterisation of particles. The microfluidic chip measures side scattered light, signal extinction and fluorescence using integrated optical fibres coupled to photomultiplier tubes. The channel is 80 μm high and 200 μm wide, and made from SU-8 patterned and sandwiched between glass substrates. Particles were focused into the analysis region using 1-D hydrodynamic focusing and typical particle velocities were 0.1 ms(-1). Excitation light is coupled into the detection channel with an optical fibre and focused into the channel using an integrated compound air lens. The electrical impedance of particles is measured at 1 MHz using micro-electrodes fabricated on the channel top and bottom. This data is used to accurately size the particles. The system is characterised using a range of different sized polystyrene beads (fluorescent and non-fluorescent). Single and mixed populations of beads were measured and the data compared with a conventional flow cytometer.     

Microfluidic droplet sorting with a high frequency ultrasound beam

This paper presents experimental results demonstrating the feasibility of high frequency ultrasonic sensing and sorting for screening single oleic acid (lipid or oil) droplets under continuous flow in a microfluidic channel. In these experiments, hydrodynamically focused lipid droplets of two different diameters (50 μm and 100 μm) are centered along the middle of the channel, which is filled with deionized (DI) water. A 30 MHz lithium niobate (LiNbO(3)) transducer, placed outside the channel, first transmits short sensing pulses to non-invasively determine the acoustic scattering properties of the individual droplets passing through the beam's focus. Integrated backscatter (IB) coefficients, utilized as a sorting criterion, are measured by analyzing the received echo signals from each droplet. When the IB values corresponding to 100 μm droplets are obtained, a custom-built LabVIEW panel commands the transducer to emit sinusoidal burst signals to commence the sorting operation. The number of droplets tested for the sorting is 139 for 50 μm droplets and 95 for 100 μm droplets. The sensing efficiencies are estimated to be 98.6% and 99.0%, respectively. The sorting is carried out by applying acoustic radiation forces to 100 μm droplets to direct them towards the upper sheath flow, thus separating them from the centered droplet flow. The sorting efficiencies are 99.3% for 50 μm droplets and 85.3% for 100 μm droplets. The results suggest that this proposed technique has the potential to be further developed into a cost-effective and efficient cell/microparticle sorting instrument.     

Quantitative distribution analysis of alumina inclusion particles in ferritic stainless steels by using laser‐induced breakdown optical emission spectrometry

This paper describes an analytical method for determining the spatial distribution of alumina inclusion particles in several ferritic stainless steels by laser‐induced breakdown optical emission spectrometry with a single‐shot laser scanned on the sample surface. For this purpose, an irradiation system, which comprised a Q‐switched Nd:YAG laser with an average energy of 50 mJ/pulse and a precisely driven X‐Y‐Z sample stage, was prepared. A Czerny–Turner‐mounting spectrograph equipped with an ICCD detector was employed for a time‐resolved measurement of the laser‐induced breakdown optical emission spectrometry signal. The intensity ratio of Al I 396.152 nm to Cr I 396.368 nm was measured each for the single shot, while the irradiation positions were step‐wise moved in the X‐Y direction and then the same sampling area was repeatedly irradiated by subsequent laser shots in the Z direction. The number of alumina particles was mapped from the intensity ratio of Al/Cr each for the irradiation points in both the lateral and in‐depth directions, enabling the distribution of alumina particles to be presented. The resolution of our measuring system was 40 μm in the lateral position and 6–7 μm in the depth direction, which were mainly determined by the crater size of a laser shot. A typical size of the alumina particles (several μm) was smaller than their resolutions; nevertheless, the suggested method would be still more effective to give the distribution of alumina particles, especially the coarse ones, because of its rapid response for the analytical result. Copyright © 2017 John Wiley & Sons, Ltd.     

Production of porous silica microparticles by membrane emulsification

A method for the production of near-monodispersed spherical silica particles with controllable porosity based on the formation of uniform emulsion droplets using membrane emulsification is described. A hydrophobic metal membrane with a 15 μm pore size and 200 μm pore spacing was used to produce near-monodispersed droplets, with a mean size that could be controlled between 65 and 240 μm containing acidified sodium silicate solution (with 4 and 6 wt % SiO(2)) in kerosene. After drying and shrinking, the final silica particles had a mean size in the range between 30 and 70 μm. The coefficient of variation for both the droplets and the particles did not exceed 35%. The most uniform particles had a mean diameter of 40 μm and coefficient of variation of 17%. By altering the pH of the sodium silicate solution and aging the gel particles in water or acetone, the internal structure of the silica particles was successfully modified, and both micro- and mesoporous near-monodispersed spherical particles were produced with an average internal pore size between 1 and 6 nm and an average surface area between 360 and 750 m(2) g(-1). A material balance and particle size analysis provided identical values for the internal voidage of the particles, when compared to the voidage as determined by BET analysis.     

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.     

Micromachined impedance spectroscopy flow cytometer for cell analysis and particle sizing

A new cytological tool, based on the microCoulter particle counter (microCPC) principle, aimed at diagnostic applications for cell counting and separation in haematology, oncology or toxicology is described. The device measures the spectral impedance of individual cells or particles and allows screening rates over 100 samples s(-1) on a single-cell basis. This analyzer is intended to drive a sorting actuator producing a subsequent cell separation. Size reduction and integration of functions are essential in achieving precise measurements and high throughput. 3D finite element simulations are presented to compare various electrode geometries and their influence on cell parameters estimation. The device is based on a glass-polyimide microfluidic chip with integrated channels and electrodes microfabricated at the length scale of the particles to be investigated (1-20 microm). A laminar liquid flow carries the suspended particles through the measurement area. Each particle's impedance signal is recorded by a differential pair of microelectrodes using the cell surrounding media as a reference. The micromachined chip and processing electronic circuit allow simultaneous impedance measurements at multiple frequencies, ranging from 100 kHz to 15 MHz. In this paper, we describe the microfabrication and characterisation of an on-chip flow-cytometer as the first building block of a complete cell-sorting device. We then discuss the signal conditioning technique and finally impedance measurements of cells and particles of different sizes and types to demonstrate the differentiation of subpopulations in a mixed sample.     

Impedance measurement technique for high-sensitivity cell detection in microstructures with non-uniform conductivity distribution

Particle detection in microstructures is a key procedure required by modern lab-on-a-chip devices. Unfortunately, state of the art approaches to impedance measuring as applied to cell detection do not perform well in regions characterized by non-homogeneous physical parameters due, for example, to the presence of air-liquid interfaces or when the particle-electrode distance is relatively high. This paper presents a robust impedance measurement technique and a circuit for detecting cells flowing in microstructures such as microchannels and microwells. Our solution makes use of an innovative three-electrode measurement scheme with asymmetric polarization in order to increase cell detection ability in microstructures featuring large electrode distances of up to 100 μm as well as to limit signal loss due to cell position relative to the electrodes. Compared to standard techniques, numerical simulations show that, with the proposed approach, the cell detection sensitivity is increased by more than 40%. In addition, we propose a custom circuit based on division instead of difference between signals, as in standard differential circuits, so as to reduce the baseline signal drift induced by non-homogeneous conductivity. A simplified analytical model shows an increase in the signal-to-noise-ratio comprised in the range 3.9-5.9. Experimental results, carried out using an open-microwell device made with flexible printed circuit board technology, are in agreement with simulations, suggesting a six-fold increase of the signal-to-noise ratio compared to the differential measurement technique. We were thus able to successfully monitor the process of isolating K562 leukemia cells inside open-microwells determining all single-cell events with no false positive detection.     

流动聚焦型微流控体系中的液滴的图案化排列和转变模式

流体在微流通道中形成剪切流场（低雷诺数）．不同于宏观体系,由于剪切力和表面张力的竞争作用,产生的液滴在微尺度下的微流通道中形成特殊的排列现象---周期性类似“晶格”排列现象．设计了新型流动聚焦型微流控芯片,分析研究在微流体系中液滴周期性图案化排列和转变机理性,液滴排列模式受两方面因素影响：水油两相的流速比值和微通道尺寸．当微通道宽度为250或300 μm时,液滴形成单层分散,双层和单层挤压排列．当微通道宽度为350 μm 时,液滴会形成单层分散到三层排列到双层挤压最后到单层挤压排列．当出口通道宽度增加到400 μm时,甚至出现了液滴四层排列的现象．同时研究了各个液滴排列模式的“转变点”.     

Newly designed flow field-flow fractionation channel for macromolecules and particles in the submicrometer and micrometer range

The flow field-flow fractionation (FIFFF) technique is a promising method for separating and analysing particles and large size macromolecules from a few nanometers to approximately 50 μm. A new fractionation channel is described featuring well defined flow conditions even for low channel heights with convenient assembling and operations features. The application of the new flow field-flow fractionation channel is proved by the analysis of pigments and other small particles of technical interest in the submicrometer range. The experimental results including multimodal size distributions are presented and discussed.     

Acoustically enriching, large-depth aquatic sampler

In marine biology, it is useful to collect water samples when exploring the distribution and diversity of microbial communities in underwater environments. In order to provide, e.g., a miniaturized submersible explorer with the capability of collecting microorganisms, a compact sample enrichment system has been developed. The sampler is 30 mm long, 15 mm wide, and just a few millimetres thick. Integrated in a multilayer steel, polyimide and glass construction is a microfluidic channel with piezoelectric transducers, where microorganism and particle samples are collected and enriched, using acoustic radiation forces for gentle and labelless trapping. High-pressure, latchable valves, using paraffin as the actuation material, at each end of the microfluidic channel keep the collected sample pristine. A funnel structure raised above the surface of the device directs water into the microfluidic channel as the vehicle propels itself or when there is a flow across its hull. The valves proved leak proof to a pressure of 2.1 MPa for 19 hours and momentary pressures of 12.5 MPa, corresponding to an ocean depth of more than 1200 metres. By reactivating the latching mechanism, small leakages through the valves could be remedied, which could thus increase the leak-less operational time. Fluorescent particles, 1.9 μm in diameter, were successfully trapped in the microfluidic channel at flow rates up to 15 μl min(-1), corresponding to an 18.5 cm s(-1) external flow rate of the sampler. In addition, liquid-suspended GFP-marked yeast cells were successfully trapped.     

Classification of cell types using a microfluidic device for mechanical and electrical measurement on single cells

This paper presents a microfluidic system for cell type classification using mechanical and electrical measurements on single cells. Cells are aspirated continuously through a constriction channel with cell elongations and impedance profiles measured simultaneously. The cell transit time through the constriction channel and the impedance amplitude ratio are quantified as cell's mechanical and electrical property indicators. The microfluidic device and measurement system were used to characterize osteoblasts (n=206) and osteocytes (n=217), revealing that osteoblasts, compared with osteocytes, have a larger cell elongation length (64.51 ± 14.98 μm vs. 39.78 ± 7.16 μm), a longer transit time (1.84 ± 1.48 s vs. 0.94 ± 1.07 s), and a higher impedance amplitude ratio (1.198 ± 0.071 vs. 1.099 ± 0.038). Pattern recognition using the neural network was applied to cell type classification, resulting in classification success rates of 69.8% (transit time alone), 85.3% (impedance amplitude ratio alone), and 93.7% (both transit time and impedance amplitude ratio as input to neural network) for osteoblasts and osteocytes. The system was also applied to test EMT6 (n=747) and EMT6/AR1.0 cells (n=770, EMT6 treated by doxorubicin) that have a comparable size distribution (cell elongation length: 51.47 ± 11.33 μm vs. 50.09 ± 9.70 μm). The effects of cell size on transit time and impedance amplitude ratio were investigated. Cell classification success rates were 51.3% (cell elongation alone), 57.5% (transit time alone), 59.6% (impedance amplitude ratio alone), and 70.2% (both transit time and impedance amplitude ratio). These preliminary results suggest that biomechanical and bioelectrical parameters, when used in combination, could provide a higher cell classification success rate than using electrical or mechanical parameter alone.     

Micro-HPLC 1: Linear “breakthrough-gradients” using the discontinuous interface between eluents in a packed gradient-generator column

A new approach is described for micro-HPLC for generating nearly linear gradients in the 40 to 550 μl volume range. These “linear” gradients have a straight line middle portion with a gentle curved onset of the gradient andd rapid attainment of the final 100% composition. Such “breakthrough-gradients” are produced by the interface between weak and strong eluent when a small (3 × 0.46 cm) packed “gradient-generator” column is switched abruptly from weak to strong eluent. The model system described here, without an analytical column, shows gradients from weak eluent (water) to strong eluent (modele with water plus acetone) using a 12-port high pressure valve to 1) vent the “flush” eluent, and simultaneously 2) begin the gradient and 3) make the sample injection. The volume of the gradient can be changed in two ways. If the breakthrough-generator is packed with very large (300 to 1600 μm nonporous particles, the gradient volume is nearly independent of the flush flow rate. The flush flow is used to quickly move the breakthrough-interface to the head of the micro-HPLC column. However, the gradient volume can be increased by using larger solid particles in the gradient-generator column because of increased eddy diffusion. If the gradient-generator is packed with porous particles (75–150 μm), the gradient volume can be changed over a broad range by simply changing the flush flow rate. This simple control over the gradient volume is due to the increased mass transfer of weak eluent out of the porous particles into the strong eluent at higher flush flows. The breakthrough-interface gradient-generator column approach provides the following advantages for micro-HPLC:

• – Gradient volumes can be readily varied over the micro-HPLC range 40–550 μl.
• – Gradient are linear with smooth and rapid onset of intial and final concentrations.
• – Gradient are produced inexpensively with a single high-pressure precision pump.
• – Gradient can be automated with a timer and single 12-port valve that provides both column regeneration and sample injection.

     

Flow-injection extraction with a microvolume module based on integrated conduits

A self-contained module for liquid—liquid extractions is described. The module contains engraved conduits for mixing of sample and ragent, an engraved segmentor, a detachable extraction coil, a membrane separator, and a rinsing system for the flow cell. The membrane in the separator is supported by a teflon-coated steel grid and can be replaced rapidly. The segmented stream enters at the centre of the circular membrane and travels through an engraved, coiled channel in contact with the membrane before leaving the membrane area at the periphery. The volume of the receptor chamber for the organic phase is 10 μl. For a detector flow-cell volume of 8 μl, an aqueous flow rate of 2.0 ml min^?1 and an organic flow rate of 1.2 ml min^?1, the “loss factors” caused by analyte dispersion are 3.2 and 1.5 for caffeine sample volumes of 40 μl and 100 μl, respectively, compared with batch extraction. The system is also tested for extraction of anionic surfactants as their ion-pairs with methylene blue.     

Sheathless elasto-inertial particle focusing and continuous separation in a straight rectangular microchannel

Particle focusing in planar geometries is essentially required in order to develop cost-effective lab-on-a-chips, such as cell counting and point-of-care (POC) devices. In this study, a novel method for sheathless particle focusing, called "Elasto-Inertial Particle Focusing", was demonstrated in a straight microchannel. The particles were notably aligned along the centerline of the straight channel under a pressure-driven flow without any additional external force or apparatus after the addition of an elasticity enhancer: PEO (poly(ethylene oxide)) (～O(100) ppm). As theoretically predicted (elasticity number: El≈O(100)), multiple equilibrium positions (centerline and corners) were observed for the viscoelastic flow without inertia, whereas three-dimensional particle focusing only occurred when neither the elasticity nor the inertia was negligible. Therefore, the three-dimensional particle focusing mechanism was attributed to the synergetic combination of the elasticity and the inertia (elasticity number: El≈O(1-10)). Furthermore, from the size dependence of the elastic force upon particles, we demonstrated that a mixture of 5.9 and 2.4 μm particles was separated at the exit of the channel in viscoelastic flows. We expect that this method can contribute to develop the miniaturized flow cytometry and microdevices for cell and particle manipulation.     

Experimental investigation of the flow induced by artificial cilia

The fluid transport produced by rectangular shaped, magnetically actuated artificial cilia of 70 μm length and 20 μm width was determined by means of phase-locked Micro Particle Image Velocimetry (μPIV) measurements in a closed microfluidic chamber. The phase-averaged flow produced by the artificial cilia reached up to 130 μm s(-1) with an actuation cycle frequency of 10 Hz. Analysis of the measured flow data indicate that the present system is capable of achieving volume flow rates of V[combining dot above](cilia) = 14 ± 4 μl min(-1) in a micro channel of 0.5 × 5 mm(2) cross-sectional area when no back pressure is built up. This corresponds to an effective pressure gradient of 6 ± 1 Pa m(-1), which equals a pressure difference of 0.6 ± 0.1 mPa over a distance of 100 μm between two rows of cilia. These results were derived analytically from the measured velocity profile by treating the cilia as a thin boundary layer. While the cilia produce phase-averaged velocities of the order of O(10(2)μm s(-1)), time-resolved measurements showed that the flow field reverses two times during one actuation cycle inducing instantaneous velocities of up to approximately 2 mm s(-1). This shows that the flow field is dominated by fluid oscillations and flow rates are expected to increase if the beating motion of the cilia is further improved.     

Integrated continuous flow polymerase chain reaction and micro-capillary electrophoresis system with bioaffinity preconcentration

An integrated and modular DNA analysis system is reported that consists of two modules: (i) A continuous flow polymerase chain reaction (CFPCR) module fabricated in a high T(g) (150°C) polycarbonate substrate in which selected gene fragments were amplified using biotin and fluorescently labeled primers accomplished by continuously shuttling small packets of PCR reagents and template through isothermal zones as opposed to heating and cooling large thermal masses typically performed in batch-type thermal reactors. (ii) μCE (micro-capillary electrophoresis) module fabricated in poly(methylmethacrylate) (PMMA), which utilized a bioaffinity selection and purification bed (2.9 μL) to preconcentrate and purify the PCR products generated from the CFPCR module prior to electrophoretic sorting. Biotin-labeled CFPCR products were hydrostatically pumped through the streptavidin-modified bed, where they were extracted onto the surface of micropillars. The affinity bed was also fabricated in PMMA and was populated with an array of microposts (50 μm width; 100 μm height) yielding a total surface area of ～117 mm(2). This solid-phase extraction (SPE) process demonstrated high selectivity for biotinylated amplicons and utilized the strong streptavidin/biotin interaction (K(d) = 10(-15) M) to generate high recoveries. The SPE selected CFPCR products were thermally denatured and single-stranded DNA released for injection into a 7-cm-long μCE channel for size-based separations and fluorescence detection. The utility of the system was demonstrated using Alu DNA typing for gender and ethnicity determinations as a model. Compared with the traditional cross-T injection procedure typically used for μCE, the affinity pre-concentration and injection procedure generated signal enhancements of 17- to 40-fold, critical for CFPCR thermal cyclers due to Taylor dispersion associated with their operation.     

Polycaprolactone solution–based ink for designing microfluidic channels on paper via 3D printing platform for biosensing application

This work reports a novel fabrication technique for development of channels on paper‐based microfluidic devices using the syringe module of a 3D printing syringe–based system. In this study, printing using polycaprolactone (PCL)‐based ink (Mw 70 000‐90 000) was employed for the generation of functional hydrophobic barriers on Whatman qualitative filter paper grade 1 (approximate thickness of 180 μm and pore diameter of 11 μm), which would effectively channelize fluid flow to multiple assay zones dedicated for different analyte detection on a microfluidic paper‐based analytical device (μPAD). The standardization studies reveal that a functional hydrophilic channel for sample conduction fabricated using the reported technique can be as narrow as 460.7 ± 20 μm and a functional hydrophobic barrier can be of any width with a lower limit of about 982.2 ± 142.75 μm when a minimum number of two layers of the ink is extruded onto paper. A comparison with the hydrodynamic model established for writing with ink is used to explain the width of the line printed by this system. A fluid flow analysis through a single channel system was also carried out to establish its conformity with the Washburn model, which governs the fluid flow in two‐dimensional μPAD. The presented fabrication technique proves to be a robust strategy that effectively taps the advantages of this 3D printing technique in the production of μPADs with enhanced speed and reproducibility.     

Rapid spatial and temporal controlled signal delivery over large cell culture areas

Controlled chemical delivery in microfluidic cell culture devices often relies on slowly evolving diffusive gradients, as the spatial and temporal control provided by fluid flow results in significant cell-perturbation. In this paper we introduce a microfluidic device architecture that allows for rapid spatial and temporal soluble signal delivery over large cell culture areas without fluid flow over the cells. In these devices the cell culture well is divided from a microfluidic channel located directly underneath the chamber by a nanoporous membrane. This configuration requires chemical signals in the microchannel to only diffuse through the thin membrane into large cell culture area, rather than diffuse in from the sides. The spatial chemical pattern within the microfluidic channel was rapidly transferred to the cell culture area with good fidelity through diffusion. The cellular temporal response to a step-function signal showed that dye reached the cell culture surface within 45 s, and achieved a static concentration in under 6 min. Chemical pulses of less than one minute were possible by temporally alternating the signal within the microfluidic channel, enabling rapid flow-free chemical microenvironment control for large cell culture areas.     

Fabrication of monolithic frits and columns for chip-based multidimensional separation devices

In this work, devices for two-dimensional separations are considered. The device contains a flow distributor, a first-dimension channel, and 17 second-dimension outlets. In the design, all connections between the first-dimension channel, the flow distributor, and the second-dimension outlets were tapered, with a minimal diameter of 20 μm. The use of photo-masking is explored for the fabrication of monolithic frits in all tapered connections. Monolithic frits with optimized permeability and length were successfully fabricated in all 33 tapered channels through light-induced polymerization, photo-masking, and selective exposure. The efficacy of the monolithic frits was demonstrated by creating a packed bed of 15-μm particles, confined within the first-dimension channel. The outlet of the first-dimension channel was successfully connected to a mass spectrometer. Effective flow confinement was demonstrated with a reversed-phase separation of a mixture of five standard peptides.