An optical counting technique with vertical hydrodynamic focusing for biological cells

A barrier in scaling laboratory processes into automated microfluidic devices has been the transfer of laboratory based assays: Where engineering meets biological protocol. One basic requirement is to reliably and accurately know the distribution and number of biological cells being dispensed. In this study, a novel optical counting technique to efficiently quantify the number of cells flowing into a microtube is presented. REH, B-lymphoid precursor leukemia, are stained with a fluorescent dye and frames of moving cells are recorded using a charge coupled device (CCD) camera. The basic principle is to calculate the total fluorescence intensity of the image and to divide it by the average intensity of a single cell. This method allows counting the number of cells with an uncertainty ±5%, which compares favorably to the standard biological methodology, based on the manual Trypan Blue assay, which is destructive to the cells and presents an uncertainty in the order of 20%. The use of a microdevice for vertical hydrodynamic focusing, which can reduce the background noise of out of focus cells by concentrating the cells in a thin layer, has further improved the technique. Computational fluid dynamics (CFD) simulation and confocal laser scanning microscopy images have shown an 82% reduction in the vertical displacement of the cells. For the flow rates imposed during this study, a throughput of 100–200 cells∕s is achieved.     

Three-dimensional cellular focusing utilizing a combination of insulator-based and metallic dielectrophoresis

Particle focusing in microfluidic devices is a necessary step in medical applications, such as detection, sorting, counting, and flow cytometry. This study proposes a microdevice that combines insulator-based and metal-electrode dielectrophoresis for the three-dimensional focusing of biological cells. Four insulating structures, which form an X pattern, are employed to confine the electric field in a conducting solution, thereby creating localized field minima in the microchannel. These electrodes, 56-μm-wide at the top and bottom surfaces, are connected to one electric pole of the power source. The electrodes connected to the opposite pole, which are at the sides of the microchannel, have one of three patterns: planar, dual-planar, or three-dimensional. Therefore, low-electric-field regions at the center of the microchannel are generated to restrain the viable HeLa cells with negative dielectrophoretic response. The array of insulating structures aforementioned is used to enhance the performance of confinement. According to numerical simulations, three-dimensional electrodes exhibit the best focusing performance, followed by dual-planar and planar electrodes. Experimental results reveal that increasing the strength of the applied electric field or decreasing the inlet flow rate significantly enhances focusing performance. The smallest width of focusing is 17 μm for an applied voltage and an inlet flow rate of 35 V and 0.5 μl/min, respectively. The effect of the inlet flow rate on focusing is insignificant for an applied voltage of 35 V. The proposed design retains the advantages of insulator-based dielectrophoresis with a relatively low required voltage. Additionally, complicated flow controls are unnecessary for the three-dimensional focusing of cells.     

Electrokinetic focusing and filtration of cells in a serpentine microchannel

Focusing cells into a single stream is usually a necessary step prior to counting and separating them in microfluidic devices such as flow cytometers and cell sorters. This work presents a sheathless electrokinetic focusing of yeast cells in a planar serpentine microchannel using dc-biased ac electric fields. The concurrent pumping and focusing of yeast cells arise from the dc electrokinetic transport and the turn-induced ac∕dc dielectrophoretic motion, respectively. The effects of electric field (including ac to dc field ratio and ac field frequency) and concentration (including buffer concentration and cell concentration) on the cell focusing performance were studied experimentally and numerically. A continuous electrokinetic filtration of E. coli cells from yeast cells was also demonstrated via their differential electrokinetic focusing in a serpentine microchannel.     

An integrated dielectrophoretic chip for continuous bioparticle filtering, focusing, sorting, trapping, and detecting

Multi-target pathogen detection using heterogeneous medical samples require continuous filtering, sorting, and trapping of debris, bioparticles, and immunocolloids within a diagnostic chip. We present an integrated AC dielectrophoretic (DEP) microfluidic platform based on planar electrodes that form three-dimensional (3D) DEP gates. This platform can continuously perform these tasks with a throughput of 3 μL∕min. Mixtures of latex particles, Escherichia coli Nissle, Lactobacillus, and Candida albicans are sorted and concentrated by these 3D DEP gates. Surface enhanced Raman scattering is used as an on-chip detection method on the concentrated bacteria. A processing rate of 500 bacteria was estimated when 100 μl of a heterogeneous colony of 10⁷ colony forming units ∕ml was processed in a single pass within 30 min.     

Frequency sweep rate dependence on the dielectrophoretic response of polystyrene beads and red blood cells

Alternating current (AC) dielectrophoresis (DEP) experiments for biological particles in microdevices are typically done at a fixed frequency. Reconstructing the DEP response curve from static frequency experiments is laborious, but essential to ascertain differences in dielectric properties of biological particles. Our lab explored the concept of sweeping the frequency as a function of time to rapidly determine the DEP response curve from fewer experiments. For the purpose of determining an ideal sweep rate, homogeneous 6.08 μm polystyrene (PS) beads were used as a model system. Translatability of the sweep rate approach to ∼7 μm red blood cells (RBC) was then verified. An Au/Ti quadrapole electrode microfluidic device was used to separately subject particles and cells to 10Vpp AC electric fields at frequencies ranging from 0.010 to 2.0 MHz over sweep rates from 0.00080 to 0.17 MHz/s. PS beads exhibited negative DEP assembly over the frequencies explored due to Maxwell-Wagner interfacial polarizations. Results demonstrate that frequency sweep rates must be slower than particle polarization timescales to achieve reliable incremental polarizations; sweep rates near 0.00080 MHz/s yielded DEP behaviors very consistent with static frequency DEP responses for both PS beads and RBCs.     

基于脉冲积累的SAR实时聚焦算法

在实际的SAR场景中,由于载机平台运动的不规律会引入相位误差,这将导致SAR图像出现模糊,甚至不能形成图像,因此需要准确地估计和补偿相位误差.提出一种较好的SAR相位历史估计算法,在方位向应用延时自相关方法进行准确的相位估计,由此实现SAR的准确聚焦成像.该相位估计方法具有较高的计算效率,非常适合于实时SAR系统.利用对实际SAR数据的聚焦处理证明了该方法的有效性.     

Isolation and enrichment of low abundant particles with insulator-based dielectrophoresis

Isolation and enrichment of low-abundant particles are essential steps in many bio-analytical and clinical applications. In this work, the capability of an insulator-based dielectrophoresis (iDEP) device for the detection and stable capture of low abundant polystyrene particles and yeast cells was evaluated. Binary and tertiary mixtures of particles and cells were tested, where the low-abundant particles had concentration ratios on the order of 1:10 000 000 compared to the other particles present in the mixture. The results demonstrated successful and stable capture and enrichment of rare particles and cells (trapping efficiencies over 99%), where particles remained trapped in a stable manner for up to 4 min. A device with four reservoirs was employed for the separation and enrichment of rare particles, where the particles of interest were first selectively concentrated and then effectively directed to a side port for future collection and analysis. The present study demonstrates that simple iDEP devices have appropriate screening capacity and can be used for handling samples containing rare particles; achieving both enrichment and isolation of low-abundant particles and cells.     

A microdevice for the creation of patent, three-dimensional endothelial cell-based microcirculatory networks

Microvascular network formation is a significant and challenging goal in the engineering of large three-dimensional artificial tissue structures. We show here the development of a fully patent, 3D endothelial cell (microvascular) microfluidic network that has a single inlet and outlet, created in only 28 h in a microdevice involving fluid flow equivalent to natural vasculature. Our microdevice features a tailored "multi-rung ladder" network, a stylized mimic of an arterial-to-venous pedicle, designed to also allow for systematic and reproducible cell seeding. Immunofluorescence staining revealed a highly contiguous endothelial monolayer (human umbilical vein endothelial cells) throughout the whole network after 24 h of continuous perfusion. This network persisted for up to 72 h of culture, providing a useful template from which the effects of surface chemistry, fluid flow, and environmental conditions on the development of artificial vascular networks ex vivo may be rapidly and robustly evaluated.     

Biomarker-free dielectrophoretic sorting of differentiating myoblast multipotent progenitor cells and their membrane analysis by Raman spectroscopy

Myoblasts are muscle derived mesenchymal stem cell progenitors that have great potential for use in regenerative medicine, especially for cardiomyogenesis grafts and intracardiac cell transplantation. To utilise such cells for pre-clinical and clinical applications, and especially for personalized medicine, it is essential to generate a synchronised, homogenous, population of cells that display phenotypic and genotypic homogeneity within a population of cells. We demonstrate that the biomarker-free technique of dielectrophoresis (DEP) can be used to discriminate cells between stages of differentiation in the C2C12 myoblast multipotent mouse model. Terminally differentiated myotubes were separated from C2C12 myoblasts to better than 96% purity, a result validated by flow cytometry and Western blotting. To determine the extent to which cell membrane capacitance, rather than cell size, determined the DEP response of a cell, C2C12 myoblasts were co-cultured with GFP-expressing MRC-5 fibroblasts of comparable size distributions (mean diameter ∼10 μm). A DEP sorting efficiency greater than 98% was achieved for these two cell types, a result concluded to arise from the fibroblasts possessing a larger membrane capacitance than the myoblasts. It is currently assumed that differences in membrane capacitance primarily reflect differences in the extent of folding or surface features of the membrane. However, our finding by Raman spectroscopy that the fibroblast membranes contained a smaller proportion of saturated lipids than those of the myoblasts suggests that the membrane chemistry should also be taken into account.     

Information requirements of cancer center researchers focusing on human biological samples and associated data

This study was undertaken to characterize the information requirements of cancer researchers who were specifically interested in human biological specimens at a comprehensive cancer center, and to determine if existing information systems could meet those needs. Information required by the cancer center researchers at the University of Pittsburgh Cancer Institute (UPCI, Pittsburgh, PA) was identified through interviews, query analysis, and analysis of publications. For topical matters, the study found that the most frequent types of questions were the following: clinical (50.18%), prognosis (17.87%), diagnosis/disorder-based (50.72%), and research-oriented (51.9%) queries. In terms of the required data elements, pathology data (17.32%) was the most frequently required, followed by clinical history and outcomes (15.18%). In addition, the study identified the 10 main questions, concerning human biological samples, and the majority of the questions were represented in a fairly discrete set of information spaces that could be well mapped into the conceptual data model created through the study. The results found in this study can be used for an initial data modeling, when creating a biomedical research data warehouse that would support the majority of the transitional research requirements of the UPCI.     

A hybrid dielectrophoretic system for trapping of microorganisms from water

Assessment of the microbial safety of water resources is among the most critical issues in global water safety. As the current detection methods have limitations such as high cost and long process time, new detection techniques have transpired among which microfluidics is the most attractive alternative. Here, we show a novel hybrid dielectrophoretic (DEP) system to separate and detect two common waterborne pathogens, Escherichia coli (E. coli), a bacterium, and Cryptosporidium parvum (C. parvum), a protozoan parasite, from water. The hybrid DEP system integrates a chemical surface coating with a microfluidic device containing inter-digitated microelectrodes to impart positive dielectrophoresis for enhanced trapping of the cells. Trimethoxy(3,3,3-trifluoropropyl) silane, (3-aminopropyl)triethoxysilane, and polydiallyl dimethyl ammonium chloride (p-DADMAC) were used as surface coatings. Static cell adhesion tests showed that among these coatings, the p-DADMAC-coated glass surface provided the most effective cell adhesion for both the pathogens. This was attributed to the positively charged p-DADMAC-coated surface interacting electrostatically with the negatively charged cells suspended in water leading to increased cell trapping efficiency. The trapping efficiency of E. coli and C. parvum increased from 29.0% and 61.3% in an uncoated DEP system to 51.9% and 82.2% in the hybrid DEP system, respectively. The hybrid system improved the cell trapping by encouraging the formation of cell pearl-chaining. The increment in trapping efficiency in the hybrid DEP system was achieved at an optimal frequency of 1 MHz and voltage of 2.5 V_pp for C. parvum and 2 V_pp for E. coli, the latter is lower than 2.5 V_pp and 7 V_pp, respectively, utilized for obtaining similar efficiency in an uncoated DEP system.     

An inexpensive microfluidic device for three-dimensional hydrodynamic focusing in imaging flow cytometry

We present design, characterization, and testing of an inexpensive, sheath-flow based microfluidic device for three-dimensional (3D) hydrodynamic focusing of cells in imaging flow cytometry. In contrast to other 3D sheathing devices, our device hydrodynamically focuses the cells in a single-file near the bottom wall of the microchannel that allows imaging cells with high magnification and low working distance objectives, without the need for small device dimensions. The relatively large dimensions of the microchannels enable easy fabrication using less-precise fabrication techniques, and the simplicity of the device design avoids the need for tedious alignment of various layers. We have characterized the performance of the device with 3D numerical simulations and validated these simulations with experiments of hydrodynamic focusing of a fluorescently dyed sample fluid. The simulations show that the width and the height of the 3D focused sample stream can be controlled independently by varying the heights of main and side channels of the device, and the flow rates of sample and sheath fluids. Based on simulations, we also provide useful guidelines for choosing the device dimensions and flow rates for focusing cells of a particular size. Thereafter, we demonstrate the applicability of our device for imaging a large number of RBCs using brightfield microscopy. We also discuss the choice of the region of interest and camera frame rate so as to image each cell individually in our device. The design of our microfluidic device makes it equally applicable for imaging cells of different sizes using various other imaging techniques such as phase-contrast and fluorescence microscopy.     

A microfluidic device for simultaneous electrical and mechanical measurements on single cells

This paper presents a microfluidic device for simultaneous mechanical and electrical characterization of single cells. The device performs two types of cellular characterization (impedance spectroscopy and micropipette aspiration) on a single chip to enable cell electrical and mechanical characterization. To investigate the performance of the device design, electrical and mechanical properties of MC-3T3 osteoblast cells were measured. Based on electrical models, membrane capacitance of MC-3T3 cells was determined to be 3.39±1.23 and 2.99±0.82 pF at the aspiration pressure of 50 and 100 Pa, respectively. Cytoplasm resistance values were 110.1±37.7 kΩ (50 Pa) and 145.2±44.3 kΩ (100 Pa). Aspiration length of cells was found to be 0.813±0.351 μm at 50 Pa and 1.771±0.623 μm at 100 Pa. Quantified Young's modulus values were 377±189 Pa at 50 Pa and 344±156 Pa at 100 Pa. Experimental results demonstrate the device's capability for characterizing both electrical and mechanical properties of single cells.     

Monitoring the dielectric response of single cells following mitochondrial adenosine triphosphate synthase inhibition by oligomycin using a dielectrophoretic cytometer

One of the main uses of adenosine triphosphate (ATP) within mammalian cells is powering the Na⁺/K⁺ ATPase pumps used to maintain ion concentrations within the cell. Since ion concentrations determine the cytoplasm conductivity, ATP concentration is expected to play a key role in controlling the cytoplasm conductivity. The two major ATP production pathways within cells are via glycolysis within the cytoplasm and via the electron transport chain within the mitochondria. In this work, a differential detector combined with dielectrophoretic (DEP) translation in a microfluidic channel was employed to observe single cell changes in the cytoplasm conductivity. The DEP response was made sensitive to changes in cytoplasm conductivity by measuring DEP response versus media conductivity and using double shell models to choose appropriate frequencies and media conductivity. Dielectric response of Chinese hamster ovary (CHO) cells was monitored following inhibition of the mitochondria ATP production by treatment with oligomycin. We show that in CHO cells following exposure to oligomycin (8 μg/ml) the cytoplasm conductivity drops, with the majority of the change occurring within 50 min. This work demonstrates that dielectric effects due to changes in ATP production can be observed at the single cell level.     

An integrated microfluidic platform to fabricate single-micrometer asymmetric giant unilamellar vesicles (GUVs) using dielectrophoretic separation of microemulsions

We present a microfluidic technique that generates asymmetric giant unilamellar vesicles (GUVs) in the size range of 2–14 μm. In our method, we (i) create water-in-oil emulsions as the precursors to build synthetic vesicles, (ii) deflect the emulsions across two oil streams containing different phospholipids at high throughput to establish an asymmetric architecture in the lipid bilayer membranes, and (iii) direct the water-in-oil emulsions across the oil–water interface of an oscillating oil jet in a co-flowing confined geometry to encapsulate the inner aqueous phase inside a lipid bilayer and complete the fabrication of GUVs. In the first step, we utilize a flow-focusing geometry with precisely controlled pneumatic pressures to form monodisperse water-in-oil emulsions. We observed different regimes in forming water-in-oil multiphase flows by changing the applied pressures and discovered a hysteretic behavior in jet breakup and droplet generation. In the second step of GUV fabrication, an oil stream containing phospholipids carries the emulsions into a separation region where we steer the emulsions across two parallel oil streams using active dielectrophoretic and pinched-flow fractionation separations. We explore the effect of applied DC voltage magnitude and carrier oil stream flow rate on the separation efficiency. We develop an image processing code that measures the degree of mixing between the two oil streams as the water-in-oil emulsions travel across them under dielectrophoretic steering to find the ideal operational conditions. Finally, we utilize an oscillating co-flowing jet to complete the formation of asymmetric giant unilamellar vesicles and transfer them to an aqueous phase. We investigate the effect of flow rates on properties of the co-flowing jet oscillating in the whipping mode (i.e., wavelength and amplitude) and define the phase diagram for the oil-in-water jet. Assays used to probe the lipid bilayer membrane of fabricated GUVs showed that membranes were unilamellar, minimal residual oil remained trapped between the two lipid leaflets, and 83% asymmetry was achieved across the lipid bilayers of GUVs.     

Microwave frequency sensor for detection of biological cells in microfluidic channels

We present details of an apparatus for capacitive detection of biomaterials in microfluidic channels operating at microwave frequencies where dielectric effects due to interfacial polarization are minimal. A circuit model is presented, which can be used to adapt this detection system for use in other microfluidic applications and to identify ones where it would not be suitable. The detection system is based on a microwave coupled transmission line resonator integrated into an interferometer. At 1.5 GHz the system is capable of detecting changes in capacitance of 650 zF with a 50 Hz bandwidth. This system is well suited to the detection of biomaterials in a variety of suspending fluids, including phosphate-buffered saline. Applications involving both model particles (polystyrene microspheres) and living cells—baker’s yeast (Saccharomyces cerevisiae) and Chinese hamster ovary cells—are presented.     

Rapid fabrication of a microdevice with concave microwells and its application in embryoid body formation

Here, we report a novel method for the fabrication of polydimethylsiloxane microdevices with complicated 3-D structures, such as concave and crater shapes, using an easily machined polymethyl methacrylate mold combined with a one-step molding process. The procedure presented here enables rapid preparation of complex 3-D microstructures varying in shape and dimensions. To regulate embryoid body (EB) formation, we fabricated a microfluidic device with an array of concave microwells and found that EBs growing in microwells maintained their shape, viability, and a high degree of homogeneity. We believe that this novel method provides an alternative for rapid prototyping, especially in fabricating devices with curved 3-D microstructures.     

A capillary dielectrophoretic chip for real-time blood cell separation from a drop of whole blood

This study proposes a capillary dielectrophoretic chip to separate blood cells from a drop of whole blood (approximately 1 μl) sample using negative dielectrophoretic force. The separating efficiency was evaluated by analyzing the image before and after dielectrophoretic force manipulation. Blood samples with various hematocrits (10%–60%) were tested with varied separating voltages and chip designs. In this study, a chip with 50 μm gap design achieved a separation efficiency of approximately 90% within 30 s when the hematocrit was in the range of 10%–50%. Furthermore, glucose concentration was electrochemically measured by separating electrodes following manipulation. The current response increased significantly (8.8-fold) after blood cell separation, which was attributed not only to the blood cell separation but also to sample disturbance by the dielectrophoretic force.