A microfabricated deformability-based flow cytometer with application to malaria

Malaria resulting from Plasmodium falciparum infection is a major cause of human suffering and mortality. Red blood cell (RBC) deformability plays a major role in the pathogenesis of malaria. Here we introduce an automated microfabricated "deformability cytometer" that measures dynamic mechanical responses of 10(3) to 10(4) individual RBCs in a cell population. Fluorescence measurements of each RBC are simultaneously acquired, resulting in a population-based correlation between biochemical properties, such as cell surface markers, and dynamic mechanical deformability. This device is especially applicable to heterogeneous cell populations. We demonstrate its ability to mechanically characterize a small number of P. falciparum-infected (ring stage) RBCs in a large population of uninfected RBCs. Furthermore, we are able to infer quantitative mechanical properties of individual RBCs from the observed dynamic behavior through a dissipative particle dynamics (DPD) model. These methods collectively provide a systematic approach to characterize the biomechanical properties of cells in a high-throughput manner.     

Microfluidic biomechanical assay for red blood cells parasitized by Plasmodium falciparum

Red blood cells parasitized by Plasmodium falciparum can be distinguished from uninfected cells and characterized on the basis of reduced deformability. To enable improved and simplified analysis, we developed a microfluidic device to measure red blood cell deformability using precisely controlled pressure. Individual red blood cells are deformed through multiple funnel-shaped constrictions with openings ranging from 5 down to 1 μm. Precisely controlled pressures are generated on-chip using a microfluidic circuit that attenuates an externally applied pressure by a factor of 100. The pressures required to squeeze each cell through the constriction are used as a readout to determine the intrinsic stiffness of each cell. Using this method, parasitized cells from ring through schizont stages were shown to be 1.5 to 200 times stiffer than uninfected cells. The measured deformability values of uninfected and parasitized cells showed clearly distinct distributions, demonstrating the potential of using this technique to study the pathophysiology of this disease, and the effect of potential drugs.     

Impedance‐based viscoelastic flow cytometry

Elastic nature of the viscoelastic fluids induces lateral migration of particles into a single streamline and can be used by microfluidic based flow cytometry devices. In this study, we investigated focusing efficiency of polyethylene oxide based viscoelastic solutions at varying ionic concentration to demonstrate their use in impedimetric particle characterization systems. Rheological properties of the viscoelastic fluid and particle focusing performance are not affected by ionic concentration. We investigated the viscoelastic focusing dynamics using polystyrene (PS) beads and human red blood cells (RBCs) suspended in the viscoelastic fluid. Elasto‐inertial focusing of PS beads was achieved with the combination of inertial and viscoelastic effects. RBCs were aligned along the channel centerline in parachute shape which yielded consistent impedimetric signals. We compared our impedance‐based microfluidic flow cytometry results for RBCs and PS beads by analyzing particle transit time and peak amplitude at varying viscoelastic focusing conditions obtained at different flow rates. We showed that single orientation, single train focusing of nonspherical RBCs can be achieved with polyethylene oxide based viscoelastic solution that has been shown to be a good candidate as a carrier fluid for impedance cytometry.     

Deformability-based cell classification and enrichment using inertial microfluidics

The ability to detect and isolate rare target cells from heterogeneous samples is in high demand in cell biology research, immunology, tissue engineering and medicine. Techniques allowing label-free cell enrichment or detection are especially important to reduce the complexity and costs towards clinical applications. Single-cell deformability has recently been recognized as a unique label-free biomarker for cell phenotype with implications for assessment of cancer invasiveness. Using a unique combination of fluid dynamic effects in a microfluidic system, we demonstrate high-throughput continuous label-free cell classification and enrichment based on cell size and deformability. The system takes advantage of a balance between deformability-induced and inertial lift forces as cells travel in a microchannel flow. Particles and droplets with varied elasticity and viscosity were found to have separate lateral dynamic equilibrium positions due to this balance of forces. We applied this system to successfully classify various cell types using cell size and deformability as distinguishing markers. Furthermore, using differences in dynamic equilibrium positions, we adapted the system to conduct passive, label-free and continuous cell enrichment based on these markers, enabling off-chip sample collection without significant gene expression changes. The presented method has practical potential for high-throughput deformability measurements and cost-effective cell separation to obtain viable target cells of interest in cancer research, immunology, and regenerative medicine.     

Fast on-demand droplet fusion using transient cavitation bubbles

A method for on-demand droplet fusion in a microfluidic channel is presented using the flow created from a single explosively expanding cavitation bubble. We test the technique for water-in-oil droplets, which are produced using a T-junction design in a microfluidic chip. The cavitation bubble is created with a pulsed laser beam focused into one droplet. High-speed photography of the dynamics reveals that the droplet fusion can be induced within a few tens of microseconds and is caused by the rapid thinning of the continuous phase film separating the droplets. The cavitation bubble collapses and re-condenses into the droplet. Droplet fusion is demonstrated for static and moving droplets, and for droplets of equal and unequal sizes. Furthermore, we reveal the diffusion dominated mixing flow and the transport of a single encapsulated cell into a fused droplet. This laser-based droplet fusion technique may find applications in micro-droplet based chemical synthesis and bioassays.     

Microfluidic Device Using a Gold Pillar Array and Integrated Electrodes for On‐chip Endothelial Cell Immobilization,Direct RBC Contact,and Amperometric Detection of Nitric Oxide

We describe a microfluidic device that can be used to detect interactions between red blood cells (RBCs) and endothelial cells using a gold pillar array (created by electrodeposition) and an integrated detection electrode. Endothelial cells can release nitric oxide (NO) via stimulation by RBC‐derived ATP. These studies incorporate on‐chip endothelial cell immobilization, direct RBC contact, and detection of NO in a single microfluidic device. In order to study the RBC‐EC interactions, this work used a microfluidic device made of a PDMS chip with two adjacent channels and a polystyrene base with embedded electrodes for creating a membrane (via gold pillars) and detecting NO (at a glassy carbon electrode coated with platinum‐black and Nafion). RBCs were pharmacologically treated with treprostinil in the absence and presence of glybenclamide, and ATP release was determined as was the resultant NO release from endothelial cells. Treprostinil treatment of RBCs resulted in ATP release that stimulated endothelial cells to release on average 1.8±0.2 nM NO per endothelial cell (average±SEM, n=8). Pretreatment of RBCs with glybenclamide inhibited treprostinil‐induced ATP release and, therefore, less NO was produced by the endothelial cells (0.92±0.1 nM NO per endothelial cell, n=7). In the future, this device can be used to study interactions between many other cell types (both adherent and non‐adherent cell lines) and incorporate other detection schemes.     

Microfluidic micropipette aspiration for measuring the deformability of single cells

We present a microfluidic technique for measuring the deformability of single cells using the pressure required to deform such cells through micrometre-scale tapered constrictions. Our technique is equivalent to whole-cell micropipette aspiration, but involves considerably simpler operation, less specialized equipment, and less technical skill. Single cells are infused into a microfluidic channel, and then deformed through a series of funnel-shaped constrictions. The constriction openings are sized to create a temporary seal with each cell as it passes through the constriction, replicating the interaction with the orifice of a micropipette. Precisely controlled deformation pressures are generated using an external source and then attenuated 100 : 1 using an on-chip microfluidic circuit. Our apparatus is capable of generating precisely controlled pressures as small as 0.3 Pa in a closed microchannel network, which is impervious to evaporative losses that normally limit the precision of such equipment. Intrinsic cell deformability, expressed as cortical tension, is determined from the threshold deformation pressure using the liquid-drop model. We measured the deformability of several types of nucleated cells and determined the optimal range of constriction openings. The cortical tension of passive human neutrophils was measured to be 37.0 ± 4.8 pN μm(-1), which is consistent with previous micropipette aspiration studies. The cortical tensions of human lymphocytes, RT4 human bladder cancer cells, and L1210 mouse lymphoma cells were measured to be 74.7 ± 9.8, 185.4 ± 25.3, and 235.4 ± 31.0 pN μm(-1) respectively. The precision and usability of our technique demonstrates its potential as a biomechanical assay for wide-spread use in biological and clinical laboratories.     

Microfluidic Cell Deformability Assay for Rapid and Efficient Kinase Screening with the CRISPR‐Cas9 System

Herein we report a CRISPR‐Cas9‐mediated loss‐of‐function kinase screen for cancer cell deformability and invasive potential in a high‐throughput microfluidic chip. In this microfluidic cell separation platform, flexible cells with high deformability and metastatic propensity flowed out, while stiff cells remained trapped. Through deep sequencing, we found that loss of certain kinases resulted in cells becoming more deformable and invasive. High‐ranking candidates identified included well‐reported tumor suppressor kinases, such as chk2, IKK‐α, p38 MAPKs, and DAPK2. A high‐ranking candidate STK4 was chosen for functional validation and identified to play an important role in the regulation of cell deformability and tumor suppression. Collectively, we have demonstrated that CRISPR‐based on‐chip mechanical screening is a potentially powerful strategy to facilitate systematic genetic analyses.     

The potential of autofluorescence for the detection of single living cells for label-free cell sorting in microfluidic systems

A novel method for studying unlabeled living mammalian cells based on their autofluorescence (AF) signal in a prototype microfluidic device is presented. When combined, cellular AF detection and microfluidic devices have the potential to facilitate high-throughput analysis of different cell populations. To demonstrate this, unlabeled cultured cells in microfluidic devices were excited with a 488 nm excitation light and the AF emission (> 505 nm) was detected using a confocal fluorescence microscope (CFM). For example, a simple microfluidic three-port glass microstructure was used together with conventional electroosmotic flow (EOF) to switch the direction of the fluid flow. As a means to test the potential of AF-based cell sorting in this microfluidic device, granulocytes were successfully differentiated from human red blood cells (RBCs) based on differences in AF. This study demonstrated the use of a simple microfabricated device to perform high-throughput live cell detection and differentiation without the need for cell-specific fluorescent labeling dyes and thereby reducing the sample preparation time. Hence, the combined use of microfluidic devices and cell AF may have many applications in single-cell analysis.     

Water cavitation of hydrogels

Using the cavitation rheology (CR) technique developed in our labs, we show that fluids with negligible interfacial tensions with a surrounding material can be used to induce an elastic, cavitation instability in that material. We do this by changing the cavitation media from air, which was demonstrated to induce cavitation at the tip of a syringe needle in previous studies, to water, which has a negligible surface tension with the surrounding poly(vinyl alcohol) hydrogel material. In this case, the critical pressure in which this instability occurs can be directly related to the elastic modulus of the surrounding network and is shown to be nearly independent of length scale. This independence of size scale has important implications in the use of CR for the characterization of mechanical properties from molecular to macroscopic length scales. © 2010 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 48: 1423–1427, 2010     

Determination of erythrocyte deformability and its correlation to cellular ATP release using microbore tubing with diameters that approximate resistance vessels in vivo

A novel method is described for measuring the deformability of red blood cells (RBCs) in tubing whose diameters approximate forces encountered in vivo. Here, RBCs from rabbits are loaded into a 50 cm section of 75 microm id microbore tubing and connected to a syringe pump. This section of tubing is then connected to a 15 cm section of 25 microm id tubing. As buffer is pumped through the flow system, the RBCs are evacuated from both sections of tubing. However, the inability of the RBCs to move freely through the 25 mirom id section of tubing results in a buildup of cells at the inlet of this portion of tubing. The continued force output by the syringe pump results in a deformation of the RBCs until all of the cells are eventually evacuated from the flow system. It was found that a measurement of the time required to reach half of the maximum pressure (1/2 P(max)) may be used as an indicator of the RBC deformability. For a given sample, a simple buffer results in less time to reach 1/2 P(max) (6.9 +/- 0.2 s) than deformable RBCs (21.6 +/- 0.8 s). To verify that the increased amount of time to reach 1/2 P(max) is indeed due to the RBCs, various hematocrits of an RBC sample were investigated and, as expected, it was found that a 12% RBC hematocrit had a higher 1/2 P(max) value (26.0 s +/- 2.2 s) when compared to a 7% hematocrit (19.1 +/- 0.3 s). In addition, RBCs chemically stiffened with glutaraldehyde were shown to be 25% less deformable than normal RBCs. Finally, a study was performed to examine the relationship between RBC deformability and ATP release and it was found that ATP release increased as a function of RBC deformability. This method greatly simplifies deformability measurements, employing only a syringe pump and microbore tubing, and may lead to a more complete understanding of the physiological significance of erythrocyte deformability.     

Cell separation based on size and deformability using microfluidic funnel ratchets

The separation of biological cells by filtration through microstructured constrictions is limited by unpredictable variations of the filter hydrodynamic resistance as cells accumulate in the microstructure. Applying a reverse flow to unclog the filter will undo the separation and reduce filter selectivity because of the reversibility of low-Reynolds number flow. We introduce a microfluidic structural ratchet mechanism to separate cells using oscillatory flow. Using model cells and microparticles, we confirmed the ability of this mechanism to sort and separate cells and particles based on size and deformability. We further demonstrate that the spatial distribution of cells after sorting is repeatable and that the separation process is irreversible. This mechanism can be applied generally to separate cells that differ based on size and deformability.     

Sonolysis of Escherichia coli and Pichia pastoris in microfluidics

We report on an efficient ultrasound based technique for lysing Escherichia coli and Pichia pastoris with oscillating cavitation bubbles in an integrated microfluidic system. The system consists of a meandering microfluidic channel and four piezoelectric transducers mounted on a glass substrate, with the ultrasound exposure and gas pressure regulated by an automatic control system. Controlled lysis of bacterial and yeast cells expressing green fluorescence protein (GFP) is studied with high-speed photography and fluorescence microscopy, and quantified with real-time polymerase chain reaction (qRT-PCR) and fluorescence intensity. The effectiveness of cell lysis correlates with the duration of ultrasound exposure. Complete lysis can be achieved within one second of ultrasound exposure with a temperature increase of less than 3.3 °C. The rod-shaped E. coli bacteria are disrupted into small fragments in less than 0.4 seconds, while the more robust elliptical P. pastoris yeast cells require around 1.0 second for complete lysis. Fluorescence intensity measurements and qRT-PCR analysis show that functionality of GFP and genomic DNA for downstream analytical assays is maintained.     

Examination of laser microbeam cell lysis in a PDMS microfluidic channel using time-resolved imaging

We use time-resolved imaging to examine the lysis dynamics of non-adherent BAF-3 cells within a microfluidic channel produced by the delivery of single highly-focused 540 ps duration laser pulses at lambda = 532 nm. Time-resolved bright-field images reveal that the delivery of the pulsed laser microbeam results in the formation of a laser-induced plasma followed by shock wave emission and cavitation bubble formation. The confinement offered by the microfluidic channel constrains substantially the cavitation bubble expansion and results in significant deformation of the PDMS channel walls. To examine the cell lysis and dispersal of the cellular contents, we acquire time-resolved fluorescence images of the process in which the cells were loaded with a fluorescent dye. These fluorescence images reveal cell lysis to occur on the nanosecond to microsecond time scale by the plasma formation and cavitation bubble dynamics. Moreover, the time-resolved fluorescence images show that while the cellular contents are dispersed by the expansion of the laser-induced cavitation bubble, the flow associated with the bubble collapse subsequently re-localizes the cellular contents to a small region. This capacity of pulsed laser microbeam irradiation to achieve rapid cell lysis in microfluidic channels with minimal dilution of the cellular contents has important implications for their use in lab-on-a-chip applications.     

New family of fluorinated polymer chips for droplet and organic solvent microfluidics

We present a new family of microfluidic chips hot embossed from a commercial fluorinated thermoplastic polymer (Dyneon THV). This material shares most of the properties of fluoro polymers (very low surface energy and resistance to chemicals), but is easier to process due to its relatively low melting point. Finally, as an elastic material it also allows easy world to chip connections. Fluoropolymer films can be imprinted by hot embossing from PDMS molds prepared by soft lithography. Chips are then sealed by an original technique (termed Monolithic-Adhesive-Bonding), using two different grades of fluoropolymer to obtain uniform mechanical, chemical and surface properties. This fabrication process is well adapted to rapid prototyping, but it also has potential for low cost industrial production, since it does not require any curing or etching step. We prepared microfluidic devices with micrometre resolution features, that are optically transparent, and that provide good resistance to pressure (up to 50 kPa). We demonstrated the transport of water droplets in fluorinated oil, and fluorescence detection of DNA within the droplets. No measurable interaction of the droplets with the channels wall was observed, alleviating the need for surface treatment previously necessary for droplet applications in microfluidic chips. These chips can also handle harsh organic solvents. For instance, we demonstrated the formation of chloroform droplets in fluorinated oil, expanding the potential for on chip microchemistry.     

Microfluidic approaches for cancer cell detection, characterization, and separation

This article reviews the recent developments in microfluidic technologies for in vitro cancer diagnosis. We summarize the working principles and experimental results of key microfluidic platforms for cancer cell detection, characterization, and separation based on cell-affinity micro-chromatography, magnetic activated micro-sorting, and cellular biophysics (e.g., cell size and mechanical and electrical properties). We examine the advantages and limitations of each technique and discuss future research opportunities for improving device throughput and purity, and for enabling on-chip analysis of captured cancer cells.     

High-throughput biophysical measurement of human red blood cells

This paper reports a microfluidic system for biophysical characterization of red blood cells (RBCs) at a speed of 100-150 cells s(-1). Electrical impedance measurement is made when single RBCs flow through a constriction channel that is marginally smaller than RBCs' diameters. The multiple parameters quantified as mechanical and electrical signatures of each RBC include transit time, impedance amplitude ratio, and impedance phase increase. Histograms, compiled from 84,073 adult RBCs (from 5 adult blood samples) and 82,253 neonatal RBCs (from 5 newborn blood samples), reveal different biophysical properties across samples and between the adult and neonatal RBC populations. In comparison with previously reported microfluidic devices for single RBC biophysical measurement, this system has a higher throughput, higher signal to noise ratio, and the capability of performing multi-parameter measurements.     

Automated analysis of single stem cells in microfluidic traps

We report a reliable strategy to perform automated image cytometry of single (non-adherent) stem cells captured in microfluidic traps. The method rapidly segments images of an entire microfluidic chip based on the detection of horizontal edges of microfluidic channels, from where the position of the trapped cells can be derived and the trapped cells identified with very high precision (>97%). We used this method to successfully quantify the efficiency and spatial distribution of single-cell loading of a microfluidic chip comprised of 2048 single-cell traps. Furthermore, cytometric analysis of trapped primary hematopoietic stem cells (HSC) faithfully recapitulated the distribution of cells in the G1 and S/G2-M phase of the cell cycle that was measured by flow cytometry. This approach should be applicable to automatically track single live cells in a wealth of microfluidic systems.     

Direct measurement of the impact of impaired erythrocyte deformability on microvascular network perfusion in a microfluidic device

The ability of red blood cells (RBCs, erythrocytes) to deform and pass through capillaries is essential for continual flow of blood in the microvasculature, which ensures an adequate supply of oxygen and nutrients, prompt removal of metabolic waste products, transport of drugs and hormones, and traffic of circulating cells to and from all living tissues. This paper presents a novel tool for evaluating the impact of impaired deformability of RBCs on the flow of blood in the microvasculature by directly measuring perfusion of a test microchannel network with dimensions and topology similar to the real microcirculation. The measurement of microchannel network perfusion is compared with RBC filtration -- a conventional assay of RBC deformability. In contrast to RBC filterability, network perfusion depends linearly on RBC deformability modulated by graded exposure to glutaraldehyde, showing a higher sensitivity to small changes of deformability. The direct measurement of microchannel network perfusion represents a new concept for the field of blood rheology and should prove beneficial for basic science and clinical applications.     

Sonoporation of suspension cells with a single cavitation bubble in a microfluidic confinement

We report here the sonoporation of HL60 (human promyelocytic leukemia) suspension cells in a microfluidic confinement using a single laser-induced cavitation bubble. Cavitation bubbles can induce membrane poration of cells located in their close vicinity. Membrane integrity of suspension cells placed in a microfluidic chamber is probed through either the calcein release out of calcein-loaded cells or the uptake of trypan blue. Cells that are located farther away than four times Rmax (maximum bubble radius) from the cavitation bubble center remain fully unaffected, while cells closer than 0.75 Rmax become porated with a probability of >75%. These results enable us to define a distance of 0.75 Rmax as a critical interaction distance of the cavitation bubble with HL60 suspension cells. These experiments suggest that flow-induced poration of suspension cells is applicable in lab-on-a-chip systems, and this might be an interesting alternative to electroporation.