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.     

Double spiral microchannel for label-free tumor cell separation and enrichment

This work reports on a passive double spiral microfluidic device allowing rapid and label-free tumor cell separation and enrichment from diluted peripheral whole blood, by exploiting the size-dependent hydrodynamic forces. A numerical model is developed to simulate the Dean flow inside the curved geometry and to track the particle/cell trajectories, which is validated against the experimental observations and serves as a theoretical foundation for optimizing the operating conditions. Results from separating tumor cells (MCF-7 and Hela) spiked into whole blood indicate that 92.28% of blood cells and 96.77% of tumor cells are collected at the inner and the middle outlet, respectively, with 88.5% tumor recovery rate at a throughput of 3.33 × 10(7) cells min(-1). We expect that this label-free microfluidic platform, driven by purely hydrodynamic forces, would have an impact on fundamental and clinical studies of circulating tumor cells.     

An integrated optofluidic platform for Raman-activated cell sorting

We report on integrated optofluidic Raman-activated cell sorting (RACS) platforms that combine multichannel microfluidic devices and laser tweezers Raman spectroscopy (LTRS) for delivery, identification, and simultaneous sorting of individual cells. The system allows label-free cell identification based on Raman spectroscopy and automated continuous cell sorting. Two optofluidic designs using hydrodynamic focusing and pinch-flow fractionation are evaluated based on their sorting design and flow velocity effect on the laser trapping efficiency at different laser power levels. A proof-of-principle demonstration of the integrated optofluidic LTRS system for the identification and sorting of two leukemia cell lines is presented. This functional prototype lays the foundation for the development of a label-free cell sorting platform based on intrinsic Raman markers for automated sampling and sorting of a large number of individual cells in solution.     

Hydrodynamic lift of vesicles and red blood cells in flow — from Fåhræus & Lindqvist to microfluidic cell sorting

Hydrodynamic lift forces acting on cells and particles in fluid flow receive ongoing attention from medicine, mathematics, physics and engineering. The early findings of Fåhræus & Lindqvist on the viscosity change of blood with the diameter of capillaries motivated extensive studies both experimentally and theoretically to illuminate the underlying physics. We review this historical development that led to the discovery of the inertial and non-inertial lift forces and elucidate the origins of these forces that are still not entirely clear. Exploiting microfluidic techniques induced a tremendous amount of new insights especially into the more complex interactions between the flow field and deformable objects like vesicles or red blood cells. We trace the way from the investigation of single cell dynamics to the recent developments of microfluidic techniques for particle and cell sorting using hydrodynamic forces. Such continuous and label-free on-chip cell sorting devices promise to revolutionize medical analyses for personalized point-of-care diagnosis. We present the state-of-the-art of different hydrodynamic lift-based techniques and discuss their advantages and limitations.     

Pinched flow coupled shear-modulated inertial microfluidics for high-throughput rare blood cell separation

Blood is a highly complex bio-fluid with cellular components making up >40% of the total volume, thus making its analysis challenging and time-consuming. In this work, we introduce a high-throughput size-based separation method for processing diluted blood using inertial microfluidics. The technique takes advantage of the preferential cell focusing in high aspect-ratio microchannels coupled with pinched flow dynamics for isolating low abundance cells from blood. As an application of the developed technique, we demonstrate the isolation of cancer cells (circulating tumor cells (CTCs)) spiked in blood by exploiting the difference in size between CTCs and hematologic cells. The microchannel dimensions and processing parameters were optimized to enable high throughput and high resolution separation, comparable to existing CTC isolation technologies. Results from experiments conducted with MCF-7 cells spiked into whole blood indicate >80% cell recovery with an impressive 3.25 × 10(5) fold enrichment over red blood cells (RBCs) and 1.2 × 10(4) fold enrichment over peripheral blood leukocytes (PBL). In spite of a 20× sample dilution, the fast operating flow rate allows the processing of ～10(8) cells min(-1) through a single microfluidic device. The device design can be easily customized for isolating other rare cells from blood including peripheral blood leukocytes and fetal nucleated red blood cells by simply varying the 'pinching' width. The advantage of simple label-free separation, combined with the ability to retrieve viable cells post enrichment and minimal sample pre-processing presents numerous applications for use in clinical diagnosis and conducting fundamental studies.     

Recent Advances in Plasmonic Nanostructures Applied for Label-free Single-cell Analysis

Cellular heterogeneity presents a major challenge in understanding the relationship between cells of particular genotype and response in disease. In order to characterize the cell-to-cell differences during the biochemical processes, single-cell analysis is necessary. Profiting from the unique localized surface plasmon resonance (LSPR) and Mie scattering, plasmonic nanostructures have revealed stable and adjustable scattering signals, avoiding photobleaching, blinking and autofluorescence phenomenon. These characterizations are propitious to the dynamic trace and biological image of single living cells. In this review, we discuss the recent advances in plasmonic nanostructures applied for label-free detection and monitoring of target cells at single-cell level by using three different techniques, surface-enhanced Raman scattering (SERS), surface-enhanced Infrared absorption spectroscopy (SEIRAS), and dark-field microscopy. Various avenues to design plasmonic probes combining spectra and imaging for single-cell analysis are demonstrated as well. We hope this review can highlight the superiority of plasmonic nanostructures in single cellular analysis, and further motivate the development of label-free cell analysis technique to elucidate cellular diversity and heterogeneity.     

Red blood cell rheology using single controlled laser-induced cavitation bubbles

The deformability of red blood cells (RBCs) is an important property that allows the cells to squeeze through small capillary vessels and can be used as an indicator for disease. We present a microfluidic based technique to quantify the deformability of RBCs by stretching a collection of RBCs on a timescale of tens of microseconds in a microfluidic chamber. This confinement constrains the motion of the cell to the imaging plane of the microscope during a transient cavitation bubble event generated with a focused and pulsed laser. We record and analyze the shape recovery of the cells with a high-speed camera and obtain a power law in time, consistent with other dynamic rheological results of RBCs. The extracted exponents are used to characterize the elastic properties of the cells. We obtain statistically significant differences of the exponents between populations of untreated RBCs and RBCs treated with two different reagents: neuraminidase reduces the cell rigidity, while wheat germ agglutinin stiffens the cell confirming previous experiments. This cavitation based technique is a candidate for high-throughput screening of elastic cell properties because many cells can be probed simultaneously in situ, thus with no pre-treatment.     

Automated cellular sample preparation using a Centrifuge-on-a-Chip

The standard centrifuge is a laboratory instrument widely used by biologists and medical technicians for preparing cell samples. Efforts to automate the operations of concentration, cell separation, and solution exchange that a centrifuge performs in a simpler and smaller platform have had limited success. Here, we present a microfluidic chip that replicates the functions of a centrifuge without moving parts or external forces. The device operates using a purely fluid dynamic phenomenon in which cells selectively enter and are maintained in microscale vortices. Continuous and sequential operation allows enrichment of cancer cells from spiked blood samples at the mL min(-1) scale, followed by fluorescent labeling of intra- and extra-cellular antigens on the cells without the need for manual pipetting and washing steps. A versatile centrifuge-analogue may open opportunities in automated, low-cost and high-throughput sample preparation as an alternative to the standard benchtop centrifuge in standardized clinical diagnostics or resource poor settings.     

Rapid detection of apoptosis in mammalian cells by using intact cell MALDI mass spectrometry

Detection of cell death has extensive applications and is of great commercial value. However, most current high-throughput cell viability assays cannot distinguish the two major forms of cell death: apoptosis and necrosis. Many apoptosis-specific detection methods exist but they are time consuming and labour intensive. In this work, we proposed a novel approach based on Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF-MS) for the specific detection of apoptosis in cultured mammalian cells. Buffer washed cells were directly mixed with a matrix solution and subsequently deposited onto the stainless steel target for MALDI analysis. The resulting mass spectrometric profiles were highly reproducible and can be used to reflect cell viability. Remarkably, the mass spectrometric profiles generated from apoptotic cells were distinct from those from either normal or necrotic cells. The apoptosis-specific features of the mass spectra were proportional to the percentage of apoptotic cells in the culture, but are independent of the drugs used to stimulate apoptosis. This is the first report on the utilization of intact cell MALDI mass spectrometry in detecting mammalian cell apoptosis, and can be used as a basis for the development of a reliable, fast, label-free and high-throughput method for detecting apoptotic cell death.     

Tracking and synchronization of the yeast cell cycle using dielectrophoretic opacity

Cell cycle synchronization is an important tool for the study of the cell division stages and signalling. It provides homogeneous cell cultures that are of importance to develop and improve processes such as protein synthesis and drug screening. The main approach today is the use of metabolic agents that block the cell cycle at a particular phase and accumulate cells at this phase, disturbing the cell physiology. We provide here a non-invasive and label-free continuous cell sorting technique to analyze and synchronize yeast cell division. By balancing opposing dielectrophoretic forces at multiple frequencies, we maximize sensitivity to the characteristic shape and internal structure changes occurring during the yeast cell cycle, allowing us to synchronize the culture in late anaphase.     

Protective effects of a freeze-dried extract of vegetables and fruits on the hydroxyl radical-mediated oxidative damage of DNA and decrease of erythrocytes deformability

The protective effects of a freeze-dried extracts of vegetables and fruits (BauYuan; BY) on the hydroxyl radical-mediated DNA strand breakages and the structural integrity of human red blood cells (RBCs) were investigated. First, the supercoiled plasmid (pEGFP-C1) DNA was subjected to oxidation damage by an ascorbate-fortified Fenton reaction and the protective effects were analyzed by agarose gel electrophoresis. In the absence of BY extracts, exposure of the high-throughput ·OH-generating system (Fe²⁺ concentration >1.0 μM) caused a complete fragmentation of DNA. Supplementation of BY extract (1 mg/mL) to the plasmid DNA prior to the exposure could prevent it significantly. In contrast, as the plasmid exposed to a low-grade ·OH-generating system (Fe²⁺<0.1 μM), the BY extract (1 mg/mL) provided an almost complete protection. Next, the cell deformabilities were measured to assess the protection effects of various BY extracts on human erythrocytes exposed to the oxidative insults. We found that both the aqueous extract and the organic solvent-derived extracts could strongly protect human RBCs from the reactive oxygen species (ROS)-mediated decrease in the deformability indices. The results implicated that the BY extracts could effectively protect the cell membrane integrity via scavenging ROS which enabling RBCs to maintain a balance of water content and surface area to prevent the drop of cell deformability.     

In search of the skeletal stem cell: isolation and separation strategies at the macro/micro scale for skeletal regeneration

Skeletal stem cells (SSCs) show great capacity for bone and cartilage repair however, current in vitro cultures are heterogeneous displaying a hierarchy of differentiation potential. SSCs represent the diminutive true multipotent stem cell fraction of bone marrow mononuclear cell (BMMNC) populations. Endeavours to isolate SSCs have generated a multitude of separation methodologies. SSCs were first identified and isolated by their ability to adhere to culture plastic. Once isolated, further separation is achieved via culture in selective or conditioned media (CM). Indeed, preferential SSC growth has been demonstrated through selective in vitro culture conditions. Other approaches have utilised cell morphology (size and shape) as selection criteria. Studies have also targeted SSCs based on their preferential adhesion to specified compounds, individually or in combination, on both macro and microscale platforms. Nevertheless, most of these methods which represent macroscale function with relatively high throughput, yield insufficient purity. Consequently, research has sought to downsize isolation methodologies to the microscale for single cell analysis. The central approach is identification of the requisite cell populations of SSC-specific surface markers that can be targeted for isolation by either positive or negative selection. SELEX and phage display technology provide apt means to sift through substantial numbers of candidate markers. In contrast, single cell analysis is the paramount advantage of microfluidics, a relatively new field for cell biology. Here cells can be separated under continuous or discontinuous flow according to intrinsic phenotypic and physicochemical properties. The combination of macroscale quantity with microscale specificity to generate robust high-throughput (HT) technology for pure SSC sorting, isolation and enrichment offers significant implications therein for skeletal regenerative strategies as a consequence of lab on chip derived methodology.     

Inertial lift enhanced phase partitioning for continuous microfluidic surface energy based sorting of particles

A new microfluidics technique that exploits the selectivity of phase partitioning and high-speed focusing capabilities of the inertial effects in flow was developed for continuous label-free sorting of particles and cells. Separations were accomplished by introducing particles at the interface of polyethylene glycol (PEG) and dextran (DEX) phases in rectangular high aspect-ratio microfluidic channels and allowing them to partition to energetically favorable locations within the PEG phase, DEX phase or interface at the center of the microchannel. Separation of partitioned particles was further enhanced via inertial lift forces that develop in high aspect-ratio microchannels that move particles to equilibrium positions close to the outer wall. Combining phase partitioning with inertial focusing ensures selectivity is possible using phase partitioning with sufficient throughput (at least an order of magnitude greater than phase partitioning alone) for application in the clinical and research setting. Using this system we accomplished separation of 15 μm polystyrene (PS) particles from 1-20 μm polymethylmethacrylate (PMMA) particles. Results confirm the feasibility of separation based on phase partitioning and enhancement of separation via inertial focusing. Approximately 86% of PS particles were isolated within the PEG phase whereas 78% of PMMA particles were isolated within the DEX phase. When a binary mixture of PS and PMMA was introduced within the device, ~83% of PS particles were isolated in the PEG phase and ~74% of PMMA particles were isolated in the DEX phase. These results confirm the feasibility of this technique for rapid and reliable separation of particles and potentially cells.     

Raman-activated cell counting for profiling carbon dioxide fixing microorganisms

Raman microspectroscopy is a label-free and nondestructive technique to measure the intrinsic chemical profile of single cells. The naturally weak Raman signals hampered the application of Raman spectroscopy for high-throughput measurements. Nearly all photosynthetic microorganisms contain carotenoids that are active molecules for resonance Raman at a 532 nm excitation wavelength. Hence, the acquisition time for a single photosynthetic microorganism can be as short as 1 ms. The carotenoid bands in Raman spectra of photosynthetic microorganisms utilizing (13)CO(2) shifted when compared to the spectra of cells utilizing (12)CO(2). Here, a mixture of (12)C- and (13)C-cyanobacterial cells were counted using a microfluidic-device-based Raman-activated cell counting procedure to prove the concept that Raman spectroscopy can be used as a high-throughput method to profile a cell population.     

Continuous cell introduction and rapid dynamic lysis for high-throughput single-cell analysis on microfludic chips with hydrodynamic focusing

A chip-based microfluidic system for high-throughput single-cell analysis is described. The system was integrated with continuous introduction of individual cells, rapid dynamic lysis, capillary electrophoretic (CE) separation and laser induced fluorescence (LIF) detection. A cross microfluidic chip with one sheath-flow channel located on each side of the sampling channel was designed. The labeled cells were hydrodynamically focused by sheath-flow streams and sequentially introduced into the cross section of the microchip under hydrostatic pressure generated by adjusting liquid levels in the reservoirs. Combined with the electric field applied on the separation channel, the aligned cells were driven into the separation channel and rapidly lysed within 33ms at the entry of the separation channel by Triton X-100 added in the sheath-flow solution. The maximum rate for introducing individual cells into the separation channel was about 150cells/min. The introduction of sheath-flow streams also significantly reduced the concentration of phosphate-buffered saline (PBS) injected into the separation channel along with single cells, thus reducing Joule heating during electrophoretic separation. The performance of this microfluidic system was evaluated by analysis of reduced glutathione (GSH) and reactive oxygen species (ROS) in single erythrocytes. A throughput of 38cells/min was obtained. The proposed method is simple and robust for high-throughput single-cell analysis, allowing for analysis of cell population with considerable size to generate results with statistical significance.     

Enhanced cell sorting and manipulation with combined optical tweezer and microfluidic chip technologies

Sorting (or isolation) and manipulation of rare cells with high recovery rate and purity are of critical importance to a wide range of physiological applications. In the current paper, we report on a generic single cell manipulation tool that integrates optical tweezers and microfluidic chip technologies for handling small cell population sorting with high accuracy. The laminar flow nature of microfluidics enables the targeted cells to be focused on a desired area for cell isolation. To recognize the target cells, we develop an image processing methodology with a recognition capability of multiple features, e.g., cell size and fluorescence label. The target cells can be moved precisely by optical tweezers to the desired destination in a noninvasive manner. The unique advantages of this sorter are its high recovery rate and purity in small cell population sorting. The design is based on dynamic fluid and dynamic light pattern, in which single as well as multiple laser traps are employed for cell transportation, and a recognition capability of multiple cell features. Experiments of sorting yeast cells and human embryonic stem cells are performed to demonstrate the effectiveness of the proposed cell sorting approach.     

Positional dependence of particles in microfludic impedance cytometry

Single cell impedance cytometry is a label-free electrical analysis method that requires minimal sample preparation and has been used to count and discriminate cells on the basis of their impedance properties. This paper shows experimental and numerically simulated impedance signals for test particles (6 μm diameter polystyrene) flowing through a microfluidic channel. The variation of impedance signal with particle position is mapped using numerical simulation and these results match closely with experimental data. We demonstrate that for a nominal 40 μm × 40 μm channel, the impedance signal is independent of position over the majority of the channel area, but shows large experimentally verifiable variation at extreme positions. The parabolic flow profile in the channel ensures that most of the sample flows through the area of uniform signal. At high flow rates inertial focusing is observed; the particles flow in equal numbers through two equilibrium positions reducing the coefficient of variance (CV) in the impedance signals to negligible values.     

Label-free, microfluidic separation and enrichment of human breast cancer cells by adhesion difference

A label-free microfluidic method for separation and enrichment of human breast cancer cells is presented using cell adhesion as a physical marker. To maximize the adhesion difference between normal epithelial and cancer cells, flat or nanostructured polymer surfaces (400 nm pillars, 400 nm perpendicular, or 400 nm parallel lines) were constructed on the bottom of polydimethylsiloxane (PDMS) microfluidic channels in a parallel fashion using a UV-assisted capillary moulding technique. The adhesion of human breast epithelial cells (MCF10A) and cancer cells (MCF7) on each channel was independently measured based on detachment assays where the adherent cells were counted with increasing flow rate after a pre-culture for a period of time (e.g., one, two, and four hours). It was found that MCF10A cells showed higher adhesion than MCF7 cells regardless of culture time and surface nanotopography at all flow rates, resulting in label-free separation and enrichment of cancer cells. For the cell types used in our study, an optimum separation was found for 2 hours pre-culture on the 400 nm perpendicular line pattern followed by flow-induced detachment at a flow rate of 200 microl min(-1). The fraction of MCF7 cells was increased from 0.36 +/- 0.04 to 0.83 +/- 0.04 under these optimized conditions.     

Modular microfluidic system fabricated in thermoplastics for the strain-specific detection of bacterial pathogens

The recent outbreaks of a lethal E. coli strain in Germany have aroused renewed interest in developing rapid, specific and accurate systems for detecting and characterizing bacterial pathogens in suspected contaminated food and/or water supplies. To address this need, we have designed, fabricated and tested an integrated modular-based microfluidic system and the accompanying assay for the strain-specific identification of bacterial pathogens. The system can carry out the entire molecular processing pipeline in a single disposable fluidic cartridge and detect single nucleotide variations in selected genes to allow for the identification of the bacterial species, even its strain with high specificity. The unique aspect of this fluidic cartridge is its modular format with task-specific modules interconnected to a fluidic motherboard to permit the selection of the target material. In addition, to minimize the amount of finishing steps for assembling the fluidic cartridge, many of the functional components were produced during the polymer molding step used to create the fluidic network. The operation of the cartridge was provided by electronic, mechanical, optical and hydraulic controls located off-chip and packaged into a small footprint instrument (1 ft(3)). The fluidic cartridge was capable of performing cell enrichment, cell lysis, solid-phase extraction (SPE) of genomic DNA, continuous flow (CF) PCR, CF ligase detection reaction (LDR) and universal DNA array readout. The cartridge was comprised of modules situated on a fluidic motherboard; the motherboard was made from polycarbonate, PC, and used for cell lysis, SPE, CF PCR and CF LDR. The modules were task-specific units and performed universal zip-code array readout or affinity enrichment of the target cells with both made from poly(methylmethacrylate), PMMA. Two genes, uidA and sipB/C, were used to discriminate between E. coli and Salmonella, and evaluated as a model system. Results showed that the fluidic system could successfully identify bacteria in <40 min with minimal operator intervention and perform strain identification, even from a mixed population with the target of a minority. We further demonstrated the ability to analyze the E. coli O157:H7 strain from a waste-water sample using enrichment followed by genotyping.     

Label-free enrichment of MCF7 breast cancer cells from leukocytes using continuous flow dielectrophoresis

Circulating tumor cells (CTCs) present in the bloodstream are strongly linked to the invasive behavior of cancer; therefore, their detection holds great significance for monitoring disease progression. Currently available CTC isolation tools are often based on tumor-specific antigen or cell size approaches. However, these techniques are limited due to the lack of a unique and universal marker for CTCs, and the overlapping size between CTCs and regular blood cells. Dielectrophoresis (DEP), governed by the intrinsic dielectric properties of the particles, is a promising marker-free, accurate, fast, and low-cost technique that enables the isolation of CTCs from blood cells. This study presents a continuous flow, antibody-free DEP-based microfluidic device to concentrate MCF7 breast cancer cells, a well-established CTC model, in the presence of leukocytes extracted from human blood samples. The enrichment strategy was determined according to the DEP responses of the corresponding cells, obtained in our previously reported DEP spectrum study. It was based on the positive-DEP integrated with hydrodynamic focusing under continuous flow. In the proposed device, the parylene microchannel with two inlets and outlets was built on top of rectangular and equally spaced isolated planar electrodes rotated certain degree relative to the main flow (13°). The recovery of MCF7 cells mixed with leukocytes was 74%–98% at a frequency of 1 MHz and a magnitude of 10–12 V_pp. Overall, the results revealed that the presented system successfully concentrates MCF7 cancer cells from leukocytes, ultimately verifying our DEP spectrum study, in which the enrichment frequency and separation strategy of the microfluidic system were determined.