Dielectrophoretic separation of platelets from diluted whole blood in microfluidic channels

The dielectrophoresis (DEP) phenomenon is used to separate platelets directly from diluted whole blood in microfluidic channels. By exploiting the fact that platelets are the smallest cell type in blood, we utilize the DEP-activated cell sorter (DACS) device to perform size-based fractionation of blood samples and continuously enrich the platelets in a label-free manner. Cytometry analysis revealed that a single pass through the two-stage DACS device yields a high purity of platelets (approximately 95%) at a throughput of approximately 2.2 x 10(4) cells/second/microchannel with minimal platelet activation. This work demonstrates gentle and label-free dielectrophoretic separation of delicate cells from complex samples and such a separation approach may open a path toward continuous screening of blood products by integrated microfluidic devices.     

Continuous separation of microparticles in a microfluidic channel via the elasto-inertial effect of non-Newtonian fluid

Pure separation and sorting of microparticles from complex fluids are essential for biochemical analyses and clinical diagnostics. However, conventional techniques require highly complex and expensive labeling processes for high purity separation. In this study, we present a simple and label-free method for separating microparticles with high purity using the elasto-inertial characteristic of a non-Newtonian fluid in microchannel flow. At the inlet, particle-containing sample flow was pushed toward the side walls by introducing sheath fluid from the center inlet. Particles of 1 μm and 5 μm in diameter, which were suspended in viscoelastic fluid, were successfully separated in the outlet channels: larger particles were notably focused on the centerline of the channel at the outlet, while smaller particles continued flowing along the side walls with minimal lateral migration towards the centerline. The same technique was further applied to separate platelets from diluted whole blood. Through cytometric analysis, we obtained a purity of collected platelets of close to 99.9%. Conclusively, our microparticle separation technique using elasto-inertial forces in non-Newtonian fluid is an effective method for separating and collecting microparticles on the basis of size differences with high purity.     

基于错流过滤原理的微流控细胞分离芯片的研制

首次提出并制备了一种错流过滤式细胞分离微流控芯片.     

Blood separation on microfluidic paper-based analytical devices

A microfluidic paper-based analytical device (μPAD) for the separation of blood plasma from whole blood is described. The device can separate plasma from whole blood and quantify plasma proteins in a single step. The μPAD was fabricated using the wax dipping method, and the final device was composed of a blood separation membrane combined with patterned Whatman No.1 paper. Blood separation membranes, LF1, MF1, VF1 and VF2 were tested for blood separation on the μPAD. The LF1 membrane was found to be the most suitable for blood separations when fabricating the μPAD by wax dipping. For blood separation, the blood cells (both red and white) were trapped on blood separation membrane allowing pure plasma to flow to the detection zone by capillary force. The LF1-μPAD was shown to be functional with human whole blood of 24-55% hematocrit without dilution, and effectively separated blood cells from plasma within 2 min when blood volumes of between 15-22 μL were added to the device. Microscopy was used to confirm that the device isolated plasma with high purity with no blood cells or cell hemolysis in the detection zone. The efficiency of blood separation on the μPAD was studied by plasma protein detection using the bromocresol green (BCG) colorimetric assay. The results revealed that protein detection on the μPAD was not significantly different from the conventional method (p > 0.05, pair t-test). The colorimetric measurement reproducibility on the μPAD was 2.62% (n = 10) and 5.84% (n = 30) for within-day and between day precision, respectively. Our proposed blood separation on μPAD has the potential for reducing turnaround time, sample volume, sample preparation and detection processes for clinical diagnosis and point-of care testing.     

A microfluidic device for separating erythrocytes polluted by lead (II) from a continuous bloodstream flow

To sort and separate erythrocytes contaminated by lead (II) from whole bloodstream flow, the first step is to use a microchannel to transport the blood cells into a microdevice. Within the device, polluted erythrocytes can be separated from the bloodstream by applying local dielectrophoretic (DEP) forces. Exploiting the fact that Pb(2+) ions attach to the membranes of the erythrocytes, we utilize the microfluidic DEP device to perform property-based fractionation of the blood samples and to separate the polluted erythrocytes from the continuous bloodstream flow. Atomic absorption spectrometer analysis reveals that, to remove lead-polluted erythrocytes, the most effective driving velocity was less than 0.1 cm/s through our microfluidic DEP device, based on an applied power of 10 V(peak-peak) and a frequency of 15.5 MHz AC field. We were able to remove 80% of the polluted erythrocytes. Using gentle DEP manipulating techniques to efficiently sort unique cells within a complex biological sample may potentially allow biological sorting to be performed outside of hospitals, in facilities without biological analyzing equipment.     

Microfluidic devices for studies of shear-dependent platelet adhesion

Adhesion of platelets to blood vessel walls is a shear stress dependent process that promotes arrest of bleeding and is mediated by the interaction of receptors expressed on platelets with various extracellular matrix (ECM) proteins that may become exposed upon vascular injury. Studies of dynamic platelet adhesion to ECM-coated substrates in conventional flow chambers require substantial fluid volumes and are difficult to perform with blood samples from a single laboratory mouse. Here we report dynamic platelet adhesion assays in two new microfluidic devices made of PDMS. Small cross-sections of the flow chambers in the devices reduce the blood volume requirements to <100 microl per assay, making the assays compatible with samples of whole blood obtained from a single mouse. One device has an array of 8 flow chambers with shear stress varying by a factor of 1.93 between adjacent chambers, covering a 100-fold range from low venous to arterial. The other device allows simultaneous high-resolution fluorescence imaging of dynamic adhesion of platelets from two different blood samples. Adhesion of platelets in the devices to three common ECM substrate coatings was verified to conform with published results. The devices were subsequently used to study the roles of extracellular and intracellular domains of integrin alphaIIbbeta3, a platelet receptor that is a central mediator of platelet aggregation and thrombus formation. The study involved wild-type mice and two genetically modified mouse strains and showed that the absence of the integrin impaired adhesion at all shear stresses, whereas a mutation in its intracellular domain reduced the adhesion only at moderate and high stresses. Because of small sample volumes required, the devices could be employed in research with genetically-modified model organisms and for adhesion tests in clinical settings with blood from neonates.     

Continuous flow microfluidic device for cell separation, cell lysis and DNA purification

A novel integrated microfluidic device that consisted of microfilter, micromixer, micropillar array, microweir, microchannel, microchamber, and porous matrix was developed to perform sample pre-treatment of whole blood. Cell separation, cell lysis and DNA purification were performed in this miniaturized device during a continuous flow process. Crossflow filtration was proposed to separate blood cells, which could successfully avoid clogging or jamming. After blood cells were lyzed in guanidine buffer, genomic DNA in white blood cells was released and adsorbed on porous matrix fabricated by anodizing silicon in HF/ethanol electrolyte. The flow process of solutions was simulated and optimized. The anodization process of porous matrix was also studied. Using the continuous flow procedure of cell separation, cell lysis and DNA adsorption, average 35.7 ng genomic DNA was purified on the integrated microfluidic device from 1 μL rat whole blood. Comparison with a commercial centrifuge method, the miniaturized device can extract comparable amounts of PCR-amplifiable DNA in 50 min. The greatest potential of this integrated miniaturized device was illustrated by pre-treating whole blood sample, where eventual integration of sample preparation, PCR, and separation on a single device could potentially enable complete detection in the fields of point-of-care genetic analysis, environmental testing, and biological warfare agent detection.     

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.     

Quantitative and qualitative analysis of a microfluidic DNA extraction system using a nanoporous AlO(x) membrane

A nanoporous aluminium oxide membrane was integrated into a microfluidic system designed to extract hgDNA (human genomic DNA) from lysed whole blood. The effectiveness of this extraction system was determined by passing known concentrations of purified hgDNA through nanoporous membranes with varying pore sizes and measuring the amount of hgDNA deposited on the membrane while also varying salt concentration in the solution. DNA extraction efficiency increased as the salt concentration increased and nanopore size decreased. Based on these results, hgDNA was extracted from whole blood while varying salt concentration, nanopore size and elution buffer to find the conditions that yield the maximum concentration of hgDNA. The optimal conditions were found to be using a low-salt lysis solution, 100 nm pores, and a cationic elution buffer. Under these conditions the combination of flow and ionic disruption were sufficient to elute the hgDNA from the membrane. The extracted hgDNA sample was analysed and evaluated using PCR (polymerase chain reaction) to determine whether the eluted sample contained PCR inhibition factors. Eluted samples from the microfluidic system were amplified without any inhibition effects. PCR using extracted samples was demonstrated for several genes of interest. This microfluidic DNA extraction system based on embedded membranes will reduce the time, space and reagents needed for DNA analysis in microfluidic systems and will prove valuable for sample preparation in lab-on-a-chip applications.     

Particle sorting using a porous membrane in a microfluidic device

Porous membranes have been fabricated based on the development of the perforated membrane mold [Y. Luo and R. N. Zare, Lab Chip, 2008, 8, 1688-1694] to create a single filter that contains multiple pore sizes ranging from 6.4 to 16.6 μm inside a monolithic three-dimensional poly(dimethylsiloxane) microfluidic structure. By overlapping two filters we are able to achieve smaller pore size openings (2.5 to 3.3 μm). This filter operates without any detectable irreversible clogging, which is achieved using a cross-flow placed in front of each filtration section. The utility of a particle-sorting device that contains this filter is demonstrated by separating polystyrene beads of different diameters with an efficiency greater than 99.9%. Additionally, we demonstrate the effectiveness of this particle-sorting device by separating whole blood samples into white blood cells and red blood cells with platelets.     

A continuous size-dependent particle separator using a negative dielectrophoretic virtual pillar array

We present a continuous size-dependent particle separator using a negative dielectrophoretic (DEP) virtual pillar array. Two major problems in the previous size-dependent particle separators include the particle clogging in the mechanical sieving structures and the fixed range of separable particle sizes. The present particle separator uses the virtual pillar array generated by negative DEP force instead of the mechanical pillar array, thus eliminating the clogging problems. It is also possible to adjust the size of separable particles since the size of virtual pillars is a function of a particle diameter, applied voltage, flow rate, etc. At an applied voltage of 500 kHz, 10 V(rms) (root mean sqaure voltage) sinusoidal wave and a flow rate of 0.40 microl min(-1), we separate 5.7 +/- 0.28 microm-, 8.0 +/- 0.80 microm-, 10.5 +/- 0.75 microm-, and 11.9 +/- 0.12 microm-diameter polystyrene (PS) beads with a separation purity of 95%, 92%, 50%, and 63%, respectively. The 10.5 microm- and 11.9 microm-diameter PS beads have relatively low separation purity of 50% and 63%. However, at an applied voltage of 8 V(rms), we separate 11.9 microm-diameter PS beads with a separation purity over 99%. At an applied voltage of 500 kHz, 10 V(rms) sinusoidal wave and a flow rate of 0.11 microl min(-1), we separate red blood cells (5.4 +/- 1.3 microm-diameter) and white blood cells (8.1 +/- 1.5 microm-diameter) with a separation purity over 99%. Therefore, the present particle separator achieves clog-free, size-dependent particle separation, which is capable of size tuning of separable particles.     

Continuous dielectrophoretic bacterial separation and concentration from physiological media of high conductivity

Biological sample processing involves purifying target analytes from various sample matrices and concentrating them to a small volume from a large volume of crude sample. This complex process is the major obstacle for developing a microfluidic diagnostic platform. In this study, we present a microfluidic device that can continuously separate and concentrate pathogenic bacterial cells from complex sample matrices such as cerebrospinal fluid and whole blood. Having overcome critical limitations of dielectrophoretic (DEP) operation in physiological media of high conductivity, we utilized target specific DEP techniques to incorporate cell separation, medium exchange, and target concentration into an integrated platform. The proposed microfluidic device can uptake mL volumes of crude biological sample and selectively concentrate target cells into a submicrolitre volume, providing ~10(4) fold of concentration. We designed the device based on the electrokinetic theory and electric field simulation, and tested the device performance with different sample types. The separation efficiency of the device was as high as 97.0% for a bead mixture in TAE buffer and 94.3% and 87.2% for E. coli in human cerebrospinal fluid and blood, respectively. A capture efficiency of 100% was achieved in the concentration chamber. With a relatively simple configuration, the proposed device provides a robust method of continuous sample processing, which can be readily integrated into a fully automated microfluidic diagnostic platform for pathogen detection and quantification.     

Multistage-multiorifice flow fractionation (MS-MOFF): continuous size-based separation of microspheres using multiple series of contraction/expansion microchannels

Previously we introduced a novel hydrodynamic method using a multi-orifice microchannel for size-based particle separation, which is called a multi-orifice flow fractionation (MOFF). The MOFF has several advantages such as continuous, non-intrusive, and minimal power consumption. However, it has a limitation that the recovery yield is relatively low. Although the recovery may be increased by adjusting parameters such as the Reynolds number and central collecting region, poor purity inevitably followed. We newly designed and fabricated a microfluidic channel for multi-stage multi-orifice flow fractionation (MS-MOFF), which is made by combining three multi-orifice segments, and consists of 3 inlets, 3 filters, 3 multi-orifice segments and 5 outlets. The structure and dimensions of the MS-MOFF were determined by the hydrodynamic principles to have constant Reynolds numbers at each multi-orifice segment. Polystyrene microspheres of two different sizes (7 μm and 15 μm) were tested. With this device, we made an attempt to improve recovery and minimize loss of purity by collecting and re-separating non-selected particles of the first separation. The final recovery successfully increased from 73.2% to 88.7% while the final purity slightly decreased from 91.4% to 89.1% (for 15 μm). These values were never achievable with the single-stage MOFF (SS-MOFF) having only one multi-orifice segment in our previous work. The MS-MOFF channel will be useful for clinical applications, such as separation of circulating tumor cells (CTC) or rare cells from human blood samples.     

A microfluidic device for continuous, real time blood plasma separation

A microfluidic device for continuous, real time blood plasma separation is introduced. The principle of the blood plasma separation from blood cells is supported by the Zweifach-Fung effect and was experimentally demonstrated using simple microchannels. The blood plasma separation device is composed of a blood inlet, a bifurcating region which leads to a purified plasma outlet, and a concentrated blood cell outlet. It was designed to separate blood plasma from an initial blood sample of up to 45% inlet hematocrit (volume percentage of cells). The microfluidic network was designed using an analogous electrical circuit, as well as analytical and numerical studies. The functionality of this device was demonstrated using defibrinated sheep blood. During 30 minutes of continuous blood infusion through the device, all the erythrocytes (red blood cells) traveled through the device toward the concentrated blood outlet while only the plasma was separated at the bifurcating regions and flowed towards the plasma outlet. The device has been operated continuously without any clogging or hemolysis of cells. The experimentally determined plasma selectivity with respect to blood hematocrit level was almost 100% regardless of the inlet hematocrit. The total plasma separation volume percent varied from 15% to 25% with increasing inlet hematocrit. Due to the device's simple structure and control mechanism, this microdevice is expected to be used for highly efficient continuous, real time cell-free blood plasma separation from blood samples for use in lab on a chip applications.     

Microfluidic system for simultaneous optical measurement of platelet aggregation at multiple shear rates in whole blood

Thrombosis is the pathological formation of platelet aggregates which occlude blood flow causing stroke and heart attack-the leading causes of death in developed nations. Instrumentation for diagnosing and exploring treatments for pathological platelet aggregation thus has the potential for major clinical impact. Most current thrombosis methods focus on single flow conditions, non-occlusive platelet adhesion, or low shear rates and so are limited in their application to comparative studies involving multiple, pathological test conditions (e.g., shear rate, stenotic geometries that mimic arteries, and rapid platelet accumulation to occlusion). The field could benefit from a low volume, high throughput, short analysis time, and low cost system while minimizing sample handling. We report on the design, fabrication, testing, and application of a microfluidic device and associated optical system for simultaneous measurement of platelet aggregation at multiple initial shear rates within four stenotic channels in label-free whole blood. Following computational design, requisite shear rates were achieved in the device by micro- surface milling a mold and subsequent PDMS casting. We applied the microfluidic system to measure platelet aggregation in whole porcine blood for shear rates spanning physiological to pathological flow conditions (500-13000 s(-1)). Real-time, non-contact, label-free, microscope-free measurements of platelet aggregation were acquired using an optical system comprising a 650 nm diode laser and a linear CCD. We observed fully occlusive platelet aggregation in less than 20 min above a threshold initial shear rate of 4000 s(-1), and no occlusive platelet aggregation below 1500 s(-1) (N = 86 trials). Accumulation of thrombus was consistent between laser intensity, light microscopy, histology, and mass flow rate measurements. The amount of blood volumes producing occlusion were dependent on shear rate. Times to occlusion were not found to be dependent on shear rate above the threshold level of 4000 s(-1). This microfluidic system enables measurement of the entire process of occlusive platelet thrombosis in whole, unlabeled blood, in vitro, at multiple shear rates. Such a system may be useful as a point-of-care diagnostic tool for studying anti-platelet therapies in individual blood samples from high-risk patients.     

Lateral-driven continuous dielectrophoretic microseparators for blood cells suspended in a highly conductive medium

This paper presents lateral-driven continuous dielectrophoretic (DEP) microseparators for separating red and white blood cells suspended in highly conductive dilute whole blood. The continuous microseparators enable the separation of blood cells based on the lateral DEP force generated by a planar interdigitated electrode array placed at an angle to the direction of flow. The simplified line charge model that we developed for the theoretical analysis was verified by comparing it with simulated and measured results. Experimental results showed that the divergent type of microseparator can continuously separate out 87.0% of the red blood cells (RBCs) and 92.1% of the white blood cells (WBCs) from dilute whole blood within 5 min simply by using a 2 MHz, 3 Vp-p AC voltage to create a gradient electric field in a medium that conducts at 17 mS cm(-1). Under the same conditions, the convergent type of microseparator could separate out 93.6% of the RBCs and 76.9% of the WBCs. We have shown that our lateral-driven continuous DEP microseparator design is practical for the continuous separation of blood cells without the need to control the conductivity of the suspension medium, overcoming critical drawbacks of DEP microseparators.     

Low-cost polymer-film spiral inertial microfluidic device for label-free separation of malignant tumor cells

We developed a low-cost polymer-film spiral inertial microfluidic device for the effective size-dependent separation of malignant tumor cells. The device was fabricated in polymer films by rapid laser cutting and chemical bonding. After fabricating the prototype device, the separation performance of our device was evaluated using particles and cells. The effects of operational flow rate, cell diameter, and cell concentration on the separation performance were explored. Our device successfully separated tumor cells from polydisperse white blood cells according to their different migration modes and lateral positions. Then, the separation of rare cells was carried out using the high-concentration lysed blood spiked with 200 tumor cells. Experimental results showed that 83.90% of the tumor cells could be recovered, while 99.87% of white blood cells could be removed. We successfully employed our device for processing clinical pleural effusion samples from patients with advanced metastatic breast cancer. Malignant tumor cells with an average purity of 2.37% could be effectively enriched, improving downstream diagnostic accuracy. Our device offers the advantages of label-free operation, low cost, and fast fabrication, thus being a potential tool for effective cell separation.     

3D Origami-based multifunction-integrated immunodevice: low-cost and multiplexed sandwich chemiluminescence immunoassay on microfluidic paper-based analytical device

A novel 3D microfluidic paper-based immunodevice, integrated with blood plasma separation from whole blood samples, automation of rinse steps, and multiplexed CL detections, was developed for the first time based on the principle of origami (denoted as origami-based device). This 3D origami-based device, comprised of one test pad surrounded by four folding tabs, could be patterned and fabricated by wax-printing on paper in bulk. In this work, a sandwich-type chemiluminescence (CL) immunoassay was introduced into this 3D origami-based immunodevice, which could separate the operational procedures into several steps including (i) folding pads above/below and (ii) addition of reagent/buffer under a specific sequence. The CL behavior, blood plasma separation, washing protocol, and incubation time were investigated in this work. The developed 3D origami-based CL immunodevice, combined with a typical luminuol-H(2)O(2) CL system and catalyzed by Ag nanoparticles, showed excellent analytical performance for the simultaneous detection of four tumor markers. The whole blood samples were assayed and the results obtained were in agreement with the reference values from the parallel single-analyte test. This paper-based microfluidic origami CL detection system provides a new strategy for a low-cost, sensitive, simultaneous multiplex immunoassay and point-of-care diagnostics.     

Highly efficient circulating tumor cell isolation from whole blood and label-free enumeration using polymer-based microfluidics with an integrated conductivity sensor

A novel microfluidic device that can selectively and specifically isolate exceedingly small numbers of circulating tumor cells (CTCs) through a monoclonal antibody (mAB) mediated process by sampling large input volumes (>/=1 mL) of whole blood directly in short time periods (<37 min) was demonstrated. The CTCs were concentrated into small volumes (190 nL), and the number of cells captured was read without labeling using an integrated conductivity sensor following release from the capture surface. The microfluidic device contained a series (51) of high-aspect ratio microchannels (35 mum width x 150 mum depth) that were replicated in poly(methyl methacrylate), PMMA, from a metal mold master. The microchannel walls were covalently decorated with mABs directed against breast cancer cells overexpressing the epithelial cell adhesion molecule (EpCAM). This microfluidic device could accept inputs of whole blood, and its CTC capture efficiency was made highly quantitative (>97%) by designing capture channels with the appropriate widths and heights. The isolated CTCs were readily released from the mAB capturing surface using trypsin. The released CTCs were then enumerated on-device using a novel, label-free solution conductivity route capable of detecting single tumor cells traveling through the detection electrodes. The conductivity readout provided near 100% detection efficiency and exquisite specificity for CTCs due to scaling factors and the nonoptimal electrical properties of potential interferences (erythrocytes or leukocytes). The simplicity in manufacturing the device and its ease of operation make it attractive for clinical applications requiring one-time use operation.     

Integrated three-dimensional filter separates nanoscale from microscale elements in a microfluidic chip

We report on the integration of a size-based three-dimensional filter, with micrometre-sized pores, in a commercial microfluidic chip. The filter is fabricated inside an already sealed microfluidic channel using the unique capabilities of two-photon polymerization. This direct-write technique enables integration of the filter by post-processing in a chip that has been fabricated by standard technologies. The filter is located at the intersection of two channels in order to control the amount of flow passing through the filter. Tests with a suspension of 3 μm polystyrene spheres in a Rhodamine 6G solution show that 100% of the spheres are stopped, while the fluorescent molecules are transmitted through the filter. We demonstrate operation up to a period of 25 minutes without any evidence of clogging. Preliminary validation of the device for plasma separation from whole blood is shown. Moreover, the filter can be cleaned and reused by reversing the flow.