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1.
Enrichment of rare cell populations such as Circulating Tumor Cells (CTCs) is a critical step before performing analysis. This paper presents a polymeric microfluidic device with integrated thick Carbon-PolyDimethylSiloxane composite (C-PDMS) electrodes designed to carry out dielectrophoretic (DEP) trapping of low abundance biological cells. Such conductive composite material presents advantages over metallic structures. Indeed, as it combines properties of both the matrix and doping particles, C-PDMS allows the easy and fast integration of conductive microstructures using a soft-lithography approach while preserving O2 plasma bonding properties of PDMS substrate and avoiding a cumbersome alignment procedure. Here, we first performed numerical simulations to demonstrate the advantage of such thick C-PDMS electrodes over a coplanar electrode configuration. It is well established that dielectrophoretic force (FDEP) decreases quickly as the distance from the electrode surface increases resulting in coplanar configuration to a low trapping efficiency at high flow rate. Here, we showed quantitatively that by using electrodes as thick as a microchannel height, it is possible to extend the DEP force influence in the whole volume of the channel compared to coplanar electrode configuration and maintaining high trapping efficiency while increasing the throughput. This model was then used to numerically optimize a thick C-PDMS electrode configuration in terms of trapping efficiency. Then, optimized microfluidic configurations were fabricated and tested at various flow rates for the trapping of MDA-MB-231 breast cancer cell line. We reached trapping efficiencies of 97% at 20 μl/h and 78.7% at 80 μl/h, for 100 μm thick electrodes. Finally, we applied our device to the separation and localized trapping of CTCs (MDA-MB-231) from a red blood cells sample (concentration ratio of 1:10).  相似文献   

2.
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.  相似文献   

3.
Isolated mitochondria display a wide range of sizes plausibly resulting from the coexistence of subpopulations, some of which may be associated with disease or aging. Strategies to separate subpopulations are needed to study the importance of these organelles in cellular functions. Here, insulator-based dielectrophoresis (iDEP) was exploited to provide a new dimension of organelle separation. The dielectrophoretic properties of isolated Fischer 344 (F344) rat semimembranosus muscle mitochondria and C57BL/6 mouse hepatic mitochondria in low conductivity buffer (0.025–0.030 S/m) at physiological pH (7.2–7.4) were studied using polydimethylsiloxane (PDMS) microfluidic devices. First, direct current (DC) and alternating current (AC) of 0–50 kHz with potentials of 0–3000 V applied over a channel length of 1 cm were separately employed to generate inhomogeneous electric fields and establish that mitochondria exhibit negative DEP (nDEP). DEP trapping potential thresholds at 0–50 kHz were also determined to be weakly dependent on applied frequency and were generally above 200 V. Second, we demonstrated a separation scheme using DC potentials <100 V to perform the first size-based iDEP sorting of mitochondria. Samples of isolated mitochondria with heterogeneous sizes (150 nm–2 μm diameters) were successfully separated into sub-micron fractions, indicating the ability to isolate mitochondria into populations based on their size.  相似文献   

4.
In this study, a 3D passivated-electrode, insulator-based dielectrophoresis microchip (3D πDEP) is presented. This technology combines the benefits of electrode-based DEP, insulator-based DEP, and three dimensional insulating features with the goal of improving trapping efficiency of biological species at low applied signals and fostering wide frequency range operation of the microfluidic device. The 3D πDEP chips were fabricated by making 3D structures in silicon using reactive ion etching. The reusable electrodes are deposited on second glass substrate and then aligned to the microfluidic channel to capacitively couple the electric signal through a 100 μm glass slide. The 3D insulating structures generate high electric field gradients, which ultimately increases the DEP force. To demonstrate the capabilities of 3D πDEP, Staphylococcus aureus was trapped from water samples under varied electrical environments. Trapping efficiencies of 100% were obtained at flow rates as high as 350 μl/h and 70% at flow rates as high as 750 μl/h. Additionally, for live bacteria samples, 100% trapping was demonstrated over a wide frequency range from 50 to 400 kHz with an amplitude applied signal of 200 Vpp. 20% trapping of bacteria was observed at applied voltages as low as 50 Vpp. We demonstrate selective trapping of live and dead bacteria at frequencies ranging from 30 to 60 kHz at 400 Vpp with over 90% of the live bacteria trapped while most of the dead bacteria escape.  相似文献   

5.
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.  相似文献   

6.
Blood analysis plays a major role in medical and science applications and white blood cells (WBCs) are an important target of analysis. We proposed an integrated microfluidic chip for direct and rapid trapping WBCs from whole blood. The microfluidic chip consists of two basic functional units: a winding channel to mix and arrays of two-layer trapping structures to trap WBCs. Red blood cells (RBCs) were eliminated through moving the winding channel and then WBCs were trapped by the arrays of trapping structures. We fabricated the PDMS (polydimethylsiloxane) chip using soft lithography and determined the critical flow velocities of tartrazine and brilliant blue water mixing and whole blood and red blood cell lysis buffer mixing in the winding channel. They are 0.25 μl/min and 0.05 μl/min, respectively. The critical flow velocity of the whole blood and red blood cell lysis buffer is lower due to larger volume of the RBCs and higher kinematic viscosity of the whole blood. The time taken for complete lysis of whole blood was about 85 s under the flow velocity 0.05 μl/min. The RBCs were lysed completely by mixing and the WBCs were trapped by the trapping structures. The chip trapped about 2.0 × 103 from 3.3 × 103 WBCs.  相似文献   

7.
The specific membrane capacitance (SMC) is an electrical parameter that correlates with both the electrical activity and morphology of the plasma membrane, which are physiological markers for cellular phenotype and health. We have developed a microfluidic device that enables impedance spectroscopy measurements of the SMC of single biological cells. Impedance spectra induced by single cells aspirated into the device are captured over a moderate frequency range (5 kHz–1 MHz). Maximum impedance sensitivity is achieved using a tapered microfluidic channel, which effectively routes electric fields across the cell membranes. The SMC is extracted by curve-fitting impedance spectra to an equivalent circuit model. From our measurement, acute myeloid leukemia (AML) cells are found to exhibit larger SMC values in hypertonic solutions as compared with those in isotonic solutions. In addition, AML cell phenotypes (AML2 and NB4) exhibiting varying metastatic potential yield distinct SMC values (AML2: 16.9 ± 1.9 mF/m2 (n = 23); NB4: 22.5 ± 4.7 mF/m2 (n = 23)). Three-dimensional finite element simulations of the microfluidic device confirm the feasibility of this approach.  相似文献   

8.
The capability of the AC dielectrophoresis (DEP) for on-chip capture and chaining of microalgae suspended in freshwaters was evaluated. The effects of freshwater composition as well as the electric field voltage, frequency, and duration, on the dielectrophoretic response of microalga Chlamydomonas reinhardtii were characterized systematically. Highest efficiency of cell alignment in one-dimensional arrays, determined by the percentage of cells in chain and the chain length, was obtained at AC-field of 20 V mm−1 and 1 kHz applied for 600 s. The DEP response and cell alignment of C. reinhardtii in water sampled from lake, pond, and river, as well as model media were affected by the chemical composition of the media. In the model media, the efficiency of DEP chaining was negatively correlated to the conductivity of the cell suspensions, being higher in suspensions with low conductivity. The cells suspended in freshwaters, however, showed anomalously high chaining at long exposure times. High concentrations of nitrate and dissolved organic matter decrease cell chaining efficiency, while phosphate and citrate concentrations increase it and favor formation of longer chains. Importantly, the application of AC-field had no effect on algal autofluorescence, cell membrane damage, or oxidative stress damages in C. reinhardtii.  相似文献   

9.
In this study, a continuous flow dielectrophoresis (DEP) microfluidic chip was fabricated and utilized to sort out the microalgae (C. vulgaris) with different lipid contents. The proposed separation scheme is to allow that the microalgae with different lipid contents experience different negative or no DEP force at the separation electrode pair under the pressure-driven flow. The microalgae that experience stronger negative DEP will be directed to the side channel while those experience less negative or no DEP force will pass through the separation electrode pair to remain in the main channel. It was found that the higher the lipid content inside the microalgae, the higher the crossover frequency. Separation of the microalgae with 13% and 21% lipid contents, and 24% and 30%–35% lipid contents was achieved at the operating frequency 7 MHz, and 10 MHz, respectively. Moreover, separation can be further verified by measurement of the fluorescence intensity of the neutral lipid inside the sorted algal cells.  相似文献   

10.
This paper presents a field-flow method for separating particle populations in a dielectrophoretic (DEP) chip with asymmetric electrodes under continuous flow. The structure of the DEP device (with one thick electrode that defines the walls of the microfluidic channel and one thin electrode), as well as the fabrication and characterization of the device, was previously described. A characteristic of this structure is that it generates an increased gradient of electric field in the vertical plane that can levitate the particles experiencing negative DEP. The separation method consists of trapping one population to the bottom of the microfluidic channel using positive DEP, while the other population that exhibits negative DEP is levitated and flowed out. Viable and nonviable yeast cells were used for testing of the separation method.  相似文献   

11.
The ability to confine flows and focus particle streams has become an integral component of the design of microfluidic systems for the analysis of a wide range of samples. Presented here is the implementation of a 3D microfluidic nozzle capable of both focusing particles as well as dynamically positioning those particles in selected flow lamina within the downstream analysis channel. Through the independent adjustment of the three sheath inlet flows, the nozzle controlled the size of a focused stream for 6, 10, and 15 μm polystyrene microparticles. Additional flow adjustment allowed the nozzle to dynamically position the focused particle stream to a specific area within the downstream channel. This unique ability provides additional capability and sample flexibility to the system. In order to gain insight into the fluidic behavior of the system, experimental conditions and results were duplicated within 4.75 μm using a COMSOL Multiphysics® model to elucidate the structure, direction, proportion, and fate of fluid lamina throughout the nozzle region. The COMSOL Multiphysics model showed that the position and distribution of particles upon entering the nozzle have negligible influence over its focusing ability, extending the experimental results into a wider range of particle sizes and system flow rates. These results are promising for the application of this design to allow for a relatively simple, fast, fully fluidically controlled nozzle for selective particle focusing and positioning for further particle analysis and sorting.  相似文献   

12.
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.  相似文献   

13.
Accurate measurement of blood viscoelasticity including viscosity and elasticity is essential in estimating blood flows in arteries, arterials, and capillaries and in investigating sub-lethal damage of RBCs. Furthermore, the blood viscoelasticity could be clinically used as key indices in monitoring patients with cardiovascular diseases. In this study, we propose a new method to simultaneously measure the viscosity and elasticity of blood by simply controlling the steady and transient blood flows in a microfluidic analogue of Wheastone-bridge channel, without fully integrated sensors and labelling operations. The microfluidic device is designed to have two inlets and outlets, two side channels, and one bridge channel connecting the two side channels. Blood and PBS solution are simultaneously delivered into the microfluidic device as test fluid and reference fluid, respectively. Using a fluidic-circuit model for the microfluidic device, the analytical formula is derived by applying the linear viscoelasticity model for rheological representation of blood. First, in the steady blood flow, the relationship between the viscosity of blood and that of PBS solution (μBlood/μPBS) is obtained by monitoring the reverse flows in the bridge channel at a specific flow-rate rate (QPBSSS/QBloodL). Next, in the transient blood flow, a sudden increase in the blood flow-rate induces the transient behaviors of the blood flow in the bridge channel. Here, the elasticity (or characteristic time) of blood can be quantitatively measured by analyzing the dynamic movement of blood in the bridge channel. The regression formula (ABlood (t) = Aα + Aβ exp [−(t − t0)/λBlood]) is selected based on the pressure difference (ΔP = PA − PB) at each junction (A, B) of both side channels. The characteristic time of blood (λBlood) is measured by analyzing the area (ABlood) filled with blood in the bridge channel by selecting an appropriate detection window in the microscopic images captured by a high-speed camera (frame rate = 200 Hz, total measurement time = 7 s). The elasticity of blood (GBlood) is identified using the relationship between the characteristic time and the viscosity of blood. For practical demonstrations, the proposed method is successfully applied to evaluate the variations in viscosity and elasticity of various blood samples: (a) various hematocrits form 20% to 50%, (b) thermal-induced treatment (50 °C for 30 min), (c) flow-induced shear stress (53 ± 0.5 mL/h for 120 min), and (d) normal rat versus spontaneously hypertensive rat. Based on these experimental demonstrations, the proposed method can be effectively used to monitor variations in viscosity and elasticity of bloods, even with the absence of fully integrated sensors, tedious labeling and calibrations.  相似文献   

14.
Isolation and enumeration of circulating tumor cells (CTCs) are used to monitor metastatic disease progression and guide cancer therapy. However, currently available technologies are limited to cells expressing specific cell surface markers, such as epithelial cell adhesion molecule (EpCAM) or have limited specificity because they are based on cell size alone. We developed a device, ApoStream™ that overcomes these limitations by exploiting differences in the biophysical characteristics between cancer cells and normal, healthy blood cells to capture CTCs using dielectrophoretic technology in a microfluidic flow chamber. Further, the system overcomes throughput limitations by operating in continuous mode for efficient isolation and enrichment of CTCs from blood. The performance of the device was optimized using a design of experiment approach for key operating parameters such as frequency, voltage and flow rates, and buffer formulations. Cell spiking studies were conducted using SKOV3 or MDA-MB-231 cell lines that have a high and low expression level of EpCAM, respectively, to demonstrate linearity and precision of recovery independent of EpCAM receptor levels. The average recovery of SKOV3 and MDA-MB-231 cancer cells spiked into approximately 12 × 106 peripheral blood mononuclear cells obtained from 7.5 ml normal human donor blood was 75.4% ± 3.1% (n = 12) and 71.2% ± 1.6% (n = 6), respectively. The intra-day and inter-day precision coefficients of variation of the device were both less than 3%. Linear regression analysis yielded a correlation coefficient (R2) of more than 0.99 for a spiking range of 4–2600 cells. The viability of MDA-MB-231 cancer cells captured with ApoStream was greater than 97.1% and there was no difference in cell growth up to 7 days in culture compared to controls. The ApoStream device demonstrated high precision and linearity of recovery of viable cancer cells independent of their EpCAM expression level. Isolation and enrichment of viable cancer cells from ApoStream enables molecular characterization of CTCs from a wide range of cancer types.  相似文献   

15.
Circulating tumor cells (CTCs) are prognostic markers for the recurrence of cancer and may carry molecular information relevant to cancer diagnosis. Dielectrophoresis (DEP) has been proposed as a molecular marker-independent approach for isolating CTCs from blood and has been shown to be broadly applicable to different types of cancers. However, existing batch-mode microfluidic DEP methods have been unable to process 10 ml clinical blood specimens rapidly enough. To achieve the required processing rates of 106 nucleated cells/min, we describe a continuous flow microfluidic processing chamber into which the peripheral blood mononuclear cell fraction of a clinical specimen is slowly injected, deionized by diffusion, and then subjected to a balance of DEP, sedimentation and hydrodynamic lift forces. These forces cause tumor cells to be transported close to the floor of the chamber, while blood cells are carried about three cell diameters above them. The tumor cells are isolated by skimming them from the bottom of the chamber while the blood cells flow to waste. The principles, design, and modeling of the continuous-flow system are presented. To illustrate operation of the technology, we demonstrate the isolation of circulating colon tumor cells from clinical specimens and verify the tumor origin of these cells by molecular analysis.  相似文献   

16.
A variety of methods have been used to introduce chemicals into a stream or to mix two or more streams of different compositions using microfluidic devices. In the following paper, the introduction of cryoprotective agents (CPAs) used during cryopreservation of cells in order to protect them from freezing injuries and increase viability post thaw is described. Dimethylsulphoxide (DMSO) is the most commonly used CPA. We aim to optimize the operating conditions of a two-stream microfluidic device to introduce a 10% vol/vol solution of DMSO into a cell suspension. Transport behavior of DMSO between two streams in the device has been experimentally characterized for a spectrum of flow conditions (0.7 < Re < 10), varying initial donor stream concentrations, (1% vol/vol < Co < 15% vol/vol) and different flow rate fractions (0.23 < fq < 0.77). The outlet cell stream concentration is analyzed for two different flow configurations: one with the cell stream flowing on top of the DMSO-rich donor stream, and the other with the cell stream flowing beneath the heavy DMSO-laden stream. We establish a transition from a diffusive mode of mass transfer to gravity-influenced convective currents for Atwood numbers (At) in the range of (1.7 × 10−3 < At < 3.1 × 10−3) for the latter configuration. Flow visualization with cells further our understanding of the effect of At on the nature of mass transport. Cell motion studies performed with Jurkat cells confirm a high cell recovery from the device while underscoring the need to collect both the streams at the outlet of the device and suggesting flow conditions that will help us achieve the target DMSO outlet concentration for clinical scale flow rates of the cell suspension.  相似文献   

17.
While advances in genomics have enabled sensitive and highly parallel detection of nucleic acid targets, the isolation and extraction of the nucleic acids remain a critical bottleneck in the workflow. We present here a simple 3D printed microfluidic chip that allows for the vortex and centrifugation free extraction of nucleic acids. This novel microfluidic chip utilizes the presence of a water and oil interface to filter out the lysate contaminants. The pure nucleic acids, while bound on cellulose particles, are magnetically moved across the oil layer. We demonstrated efficient and rapid extraction of spiked Human Papillomavirus (HPV) 18 plasmids in specimen transport medium, in under 15 min. An overall extraction efficiency of 61% is observed across a range of HPV plasmid concentrations (5 × 101 to 5 × 106 copies/100 μl). The magnetic, interfacial, and viscous drag forces inside the microgeometries of the chip are modeled. We have also developed a kinetics model for the adsorption of nucleic acids on cellulose functionalized superparamagnetic beads. We also clarify here the role of carrier nucleic acids in the adsorption and isolation of nucleic acids. Based on the various mechanistic insights detailed here, customized microfluidic devices can be designed to meet the range of current and emerging point of care diagnostics needs.  相似文献   

18.
Lewpiriyawong N  Yang C 《Biomicrofluidics》2012,6(1):12807-128079
The recent development of microfluidic “lab on a chip” devices requires the need to continuously separate submicron particles. Here, we present a PDMS microfluidic device with sidewall conducting PDMS (AgPDMS) composite electrodes capable of separating submicron particles in hydrodynamic flow. In particular, the device can service dual functions. First, the AgPDMS composite electrodes embedded in a sidewall of the device channel allow for performing AC-dielectrophoretic (DEP) characterization through direct microscopic observation of particle behavior. Characterization experiments are carried out for numerous parameters including particle size, medium conductivity, and AC field frequency to reveal important dielectrophoresis DEP information in terms of the crossover frequency and positive/negative DEP behavior under specific frequencies. Second, the device offers an advantage that sidewall AgPDMS composite electrodes can produce strong DEP effects throughout the entire channel height, and thus the robustness of the on-chip particle separation is demonstrated for continuous separation in a flowing mixture of 0.5 and 5 μm particles with 100% separation efficiency.  相似文献   

19.
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.  相似文献   

20.
This paper describes an integrated microfluidic chip that is capable of rapidly and quantitatively measuring the concentration of a bladder cancer biomarker, apolipoprotein A1, in urine samples. All of the microfluidic components, including the fluid transport system, the micro-valve, and the micro-mixer, were driven by negative pressure, which simplifies the use of the chip and facilitates commercialization. Magnetic beads were used as a solid support for the primary antibody, which captured apolipoprotein A1 in patients'' urine. Because of the three-dimensional structure of the magnetic beads, the concentration range of the target that could be detected was as high as 2000 ng ml−1. Because this concentration is 100 times higher than that quantifiable using a 96-well plate with the same enzyme-linked immunosorbent assay (ELISA) kit, the dilution of the patient''s urine can be avoided or greatly reduced. The limit of detection was determined to be approximately 10 ng ml−1, which is lower than the cutoff value for diagnosing bladder cancer (11.16 ng ml−1). When the values measured using the microfluidic chip were compared with those measured using conventional ELISA using a 96-well plate for five patients, the deviations were 0.9%, 6.8%, 9.4%, 1.8%, and 5.8%. The entire measurement time is 6-fold faster than that of conventional ELISA. This microfluidic device shows significant potential for point-of-care applications.  相似文献   

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