Microfluidic continuous magnetophoretic protein separation using nanoparticle aggregates

We demonstrate a microfluidic continuous-flow protein separation process in which silica-coated superparamagnetic nanoparticles interact preferentially with hemoglobin in a mixture with bovine serum albumin, and the resulting hemoglobin-nanoparticle aggregates are recovered online using magnetophoresis. We present detailed modeling and analysis of this process yielding quantitative estimates of the recovery of both proteins, validated by experiments. While several previous studies utilize an average particle size in modeling magnetophoretic particle trajectories or process design, in this study we emphasize the importance of accounting for particle size distributions in calculating particle recovery, and therefore in estimating separation efficiency. We combine experimentally measured size distributions of protein-nanoparticle aggregates with simulations of particle trajectories and provide a simple analytical method to calculate the efficiency of separation at various flow speeds, which fully accounts for heterogeneity in particle sizes. Our method can potentially be used for affinity based biomolecular separations at both analytical and preparative scales by exploiting well-established techniques to functionalize nanoparticle surfaces with selective ligands. Further, the modeling methodology presented here may be applied to provide better estimates of particle recovery in a broad range of magnetophoretic separation processes involving heterogeneity in particle sizes.     

Diamagnetic capture mode magnetophoretic microseparator for blood cells

This paper presents the characterization of a continuous diamagnetic capture (DMC) mode magnetophoretic microseparator for separating red and white blood cells from diluted whole blood based on their native magnetic properties. The DMC microseparator separated the blood cells using a high-gradient magnetic separation (HGMS) method without the use of additives such as magnetic beads. The microseparator was fabricated using microfabrication technology, enabling the integration of microscale magnetic flux concentrators in an aqueous microenvironment. Experimental results show that the DMC microseparator can continuously separate out 89.7% of red blood cells (RBCs) from diluted whole blood within 5 min using an external magnetic flux of 0.2 T from a permanent magnet. Monitoring white blood cells (WBCs) probed with a fluorescence dye show that 72.7% of WBCs were separated out within 10 min in the DMC microseparator using a 0.2 T external applied magnetic flux. Consequently, the DMC microseparator may facilitate the separation of WBCs from whole blood in applications such as a genetic sample preparation and blood borne disease detection. [1574].     

Microfluidic device with dual-channel fluorescence acquisition for quantification/identification of cancer cells

This paper presents an optofluidic device for cell discrimination with two independent interrogation regions. Pumping light is coupled to the device, and cell fluorescence is extracted from the two interrogation zones by using optical fibers embedded in the optofluidic chip. To test the reliability of this device, AU-565 cells—expressing EpCAM and HER2 receptors—and RAMOS cells were mixed in a controlled manner, confined inside a hydrodynamic focused flow in the microfluidic chip and detected individually so that they could be discriminated as positive (signal reception from fluorescently labeled antibodies from the AU-565 cells) or negative events (RAMOS cells). A correlation analysis of the two signals reduces the influence of noise on the overall data.     

Microfluidic system for extraterrestrial artificial photosynthetic device

Microsystem Technologies - In situ resource utilization (ISRU) is an important way to provide oxygen and fuel for human survival in future extraterrestrial exploration. At present, the main ways of...     

Microfluidic depletion of red blood cells from whole blood in high-aspect-ratio microchannels

A method for the depletion of red blood cells (RBCs) from whole blood at high volume flow rates is proposed and experimentally investigated. The approach exploits cell-screening effects at microchannel intersections with well-adjusted flow rates. It mimics blood flow phenomena previously observed and characterized in the microvascular system of living organisms. Because of the purely hydrodynamic nature of the depletion mechanism, the structural features on the device can be significantly larger than the cell dimensions in contrast to micromachined filter devices based on physical retention of cells/particles. Consequently, device fabrication is relatively straightforward and inexpensive. Cell depleted liquid can be withdrawn from the device in a continuous operation mode, thus avoiding the principal limitation of finite filter capacity associated with size exclusion based approaches. The use of high-aspect-ratio channels allowed for a combination of both cell screening action and high fluidic throughput in the ml/min regime. The experimental data relating flow velocities, channel dimension, cell depletion efficiency, and overall yield can be qualitatively interpreted using an adapted theoretical model originally developed by Fenton et al. Eventually, the method could serve as a simple, highly versatile pre-analytical sample preparation module for the manipulation of the particle density of suspensions in a miniaturized total analysis system.     

Microfluidic counterflow centrifugal elutriation system for sedimentation-based cell separation

We present a centrifugal microfluidic system for precise cell/particle sorting using the concept of counterflow centrifugal elutriation (CCE). A conventional CCE system uses a rotor device incorporating a flow-through separation chamber, in which the balance of centrifugal and counterflow drag forces exerted on particles is gradually shifted by changing the flow rate and/or the rotation speed. In the present system, both the centrifugal and the fluid forces are generated through microdevice rotation in order to significantly simplify the setup of the conventional CCE. In addition, the density gradient of the medium is employed to elute particles/cells of different sedimentation velocities stepwise from the separation chamber instead of changing the rotation speed. We successfully separated polymer particles with diameters of 1.0–5.0 μm using a branched loading channel for focusing particles to the center of the separation chamber. We also demonstrated the sorting of blood cells for biological applications. This system may provide a versatile means for cell/particle sorting in a general biological laboratory and function as a unit operation in various centrifugal microfluidic platforms for biochemical experiments and clinical diagnosis.     

Computational and performance analysis of a continuous magnetophoretic bioseparation chip with alternating magnetic fields

This paper presents the modeling and optimization of a magnetophoretic bioseparation chip for isolating cells, such as circulating tumor cells from the peripheral blood. The chip consists of a continuous-flow microfluidic platform that contains locally engineered magnetic field gradients. The high-gradient magnetic field produced by the magnets is spatially non-uniform and gives rise to an attractive force on magnetic particles flowing through a fluidic channel. Simulations of the particle–fluid transport and the magnetic force are performed to predict the trajectories and capture lengths of the particles within the fluidic channel. The computational model takes into account key forces, such as the magnetic and fluidic forces and their effect on design parameters for an effective separation. The results show that the microfluidic device has the capability of separating various cells from their native environment. An experimental study is also conducted to verify and validate the simulation results. Finally, to improve the performance of the separation device, a parametric study is performed to investigate the effects of the magnetic bead size, cell size, number of beads per cell, and flow rate on the cell separation performance.     

Microfluidic bioreactors for culture of non-adherent cells

Microfluidic bioreactors (μBR) are becoming increasingly popular for cell culture, sample preparation and analysis in case of routine genetic and clinical diagnostics. We present a novel μBR for non-adherent cells designed to mimic in vivo perfusion of cells based on diffusion of media through a sandwiched membrane. The culture chamber and perfusion chamber are separated by a sandwiched membrane and each chamber has separate inlet/outlets for easy loading/unloading of cells and perfusion of the media. The perfusion of media and exchange of nutrients occur through the sandwiched membrane, which was also verified with simulations. Finally, we present the application of this device for cytogenetic sample preparation, whereby we culture and arrest peripheral T-lymphocytes in metaphase and later fix them in the μBR. The expansion of T-lymphocytes from an unknown patient sample was quantified by means of CFSE staining and subsequent counting in a flow cytometer. To conclude on the applicability of μBR for genetic diagnostics, we prepare chromosome spreads on glass slides from the cultured samples, which is the primary step for metaphase FISH analysis.     

High throughput blood plasma separation using a passive PMMA microfluidic device

Since plasma is rich in many biomarkers used in clinical diagnostic experiments, microscale blood plasma separation is a primitive step in most of microfluidic analytical chips. In this paper, a passive microfluidic device for on-chip blood plasma separation based on Zweifach–Fung effect and plasma skimming was designed and fabricated by hot embossing of microchannels on a PMMA substrate and thermal bonding process. Human blood was diluted in various times and injected into the device. The main novelty of the proposed microfluidic device is the design of diffuser-shaped daughter channels. Our results demonstrated that this design exerted a considerable positive influence on the separation efficiency of the passive separator device, and the separation efficiency of 66.6 % was achieved. The optimum purity efficiency of 70 % was achieved for 1:100 dilution times.     

Microfluidic network-based combinatorial dilution device for high throughput screening and optimization

We present a combinatorial dilution device using a three-layer microfluidic network that can produce systematic variations of buffer and additive solutions in a combinatorial fashion for high throughput screening and optimization. A proof-of-concept device providing seven combinations (ABC/D, AB/D, BC/D, AC/D, A/D, B/D, and C/D) of three additive samples (A, B, and C) into a buffer solution (D) has been demonstrated. Such combinations are often used in simplex-centroid mixture DOE (design of experiments), useful techniques to minimize the experimental efforts at maximal information output with systematic variations of large-scale components. Based on mathematical and electrical modeling and computational fluid dynamic simulation, the device has been designed, fabricated, and characterized.     

Microfluidic reactor array device for massively parallel in situ synthesis of oligonucleotides

We have designed and fabricated a microfluidic reactor array device for massively parallel in situ synthesis of oligonucleotides (oDNA). The device is made of glass anodically bonded to silicon consisting of three level features: microreactors, microchannels and through inlet/outlet holes. Main challenges in the design of this device include preventing diffusion of photogenerated reagents upon activation and achieving uniform reagent flow through thousands of parallel reactors. The device embodies a simple and effective dynamic isolation mechanism which prevents the intermixing of active reagents between discrete microreactors. Depending on the design parameters, it is possible to achieve uniform flow and synthesis reaction in all of the reactors by proper design of the microreactors and the microchannels. We demonstrated the use of this device on a solution-based, light-directed parallel in situ oDNA synthesis. We were able to synthesize long oDNA, up to 120 mers at stepwise yield of 98%. The quality of our microfluidic oDNA microarray including sensitivity, signal noise, specificity, spot variation and accuracy was characterized. Our microfluidic reactor array devices show a great potential for genomics and proteomics researches.     

Design of microfluidic channels for magnetic separation of malaria-infected red blood cells

This study is motivated by the development of a blood cell filtration device for removal of malaria-infected, parasitized red blood cells (pRBCs). The blood was modeled as a multi-component fluid using the computational fluid dynamics discrete element method (CFD-DEM), wherein plasma was treated as a Newtonian fluid and the red blood cells (RBCs) were modeled as soft-sphere solid particles which move under the influence of drag, collisions with other RBCs, and a magnetic force. The CFD-DEM model was first validated by a comparison with experimental data from Han and Frazier (Lab Chip 6:265–273, 2006) involving a microfluidic magnetophoretic separator for paramagnetic deoxygenated blood cells. The computational model was then applied to a parametric study of a parallel-plate separator having hematocrit of 40 % with 10 % of the RBCs as pRBCs. Specifically, we investigated the hypothesis of introducing an upstream constriction to the channel to divert the magnetic cells within the near-wall layer where the magnetic force is greatest. Simulations compared the efficacy of various geometries upon the stratification efficiency of the pRBCs. For a channel with nominal height of 100 µm, the addition of an upstream constriction of 80 % improved the proportion of pRBCs retained adjacent to the magnetic wall (separation efficiency) by almost twofold, from 26 to 49 %. Further addition of a downstream diffuser reduced remixing and hence improved separation efficiency to 72 %. The constriction introduced a greater pressure drop (from 17 to 495 Pa), which should be considered when scaling up this design for a clinical-sized system. Overall, the advantages of this design include its ability to accommodate physiological hematocrit and high throughput, which is critical for clinical implementation as a blood-filtration system.     

Microfluidic separation of magnetic particles with soft magnetic microstructures

This paper demonstrates simple and cost-effective microfluidic devices for enhanced separation of magnetic particles by using soft magnetic microstructures. By injecting a mixture of iron powder and polydimethylsiloxane (PDMS) into a prefabricated channel, an iron–PDMS microstructure was fabricated next to a microfluidic channel. Placed between two external permanent magnets, the magnetized iron–PDMS microstructure induces localized and strong forces on the magnetic particles in the direction perpendicular to the fluid flow. Due to the small distance between the microstructure and the fluid channel, the localized large magnetic field gradients result a vertical force on the magnetic particles, leading to enhanced separation of the particles. Numerical simulations were developed to compute the particle trajectories and agreed well with experimental data. Systematic experiments and numerical simulation were conducted to study the effect of relevant factors on the transport of superparamagnetic particles, including the shape of iron–PDMS microstructure, mass ratio of iron–PDMS composite, width of the microfluidic channel, and average flow velocity.     

Microfluidic device for the combinatorial application and maintenance of dynamically imposed diffusional gradients

This article reports a microfluidic device for the generation of stable, steady-state, user-defined concentration profiles for the long-term maintenance of stable diffusion gradients. The microinstrument allows both dynamic temporal and dynamic spatial control over user-defined concentrations and concentration gradients of multiple chemicals. With this device, one can create an in vitro environment capable of approximating the complex in vivo biological condition for cellular studies. In addition, the device has potential application in combinatory drug discovery, electrophoretic applications, ligand binding, etc. 3D computer simulations and analysis of arbitrary concentration profiles are presented along with experimental validation using multiple diffusing species.     

Microfluidic device for robust generation of two-component liquid-in-air slugs with individually controlled composition

Using liquid slugs as microreactors and microvessels enable precise control over the conditions of their contents on short-time scales for a wide variety of applications. Particularly for screening applications, there is a need for control of slug parameters such as size and composition. We describe a new microfluidic approach for creating slugs in air, each comprising a size and composition that can be selected individually for each slug. Two-component slugs are formed by first metering the desired volume of each reagent, merging the two volumes into an end-to-end slug, and propelling the slug to induce mixing. Volume control is achieved by a novel mechanism: two closed chambers on the chip are initially filled with air, and a valve in each is briefly opened to admit one of the reagents. The pressure of each reagent can be individually selected and determines the amount of air compression, and thus the amount of liquid that is admitted into each chamber. We describe the theory of operation, characterize the slug generation chip, and demonstrate the creation of slugs of different compositions. The use of microvalves in this approach enables robust operation with different liquids, and also enables one to work with extremely small samples, even down to a few slug volumes. The latter is important for applications involving precious reagents such as optimizing the reaction conditions for radiolabeling biological molecules as tracers for positron emission tomography. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10404-010-0617-0) contains supplementary material, which is available to authorized users.     

A tunable microfluidic-based filter modulated by pneumatic pressure for separation of blood cells

This article presents a new microfluidic-based filter for the separation of microbeads or blood cells with a high filtration rate. The device was composed of a circular micropump for automatic liquid transport, and a normally closed valve located at the filter zone for separation of beads or cells. The filtration mechanism was based on the tunable deformation of polydimethylsiloxane (PDMS) membranes that defined the gap between a floating block structure and the substrate which determined the maximum diameter of the beads/cells that can pass through the filter. Another unique feature of this filter is an unclogging mechanism using a suction force, resulting in a back flow to remove any trapped beads/cells in the filter zone when the PDMS membrane was restored to its initial state. The separation performance of the proposed device was first experimentally evaluated by using microbeads. The results showed that this device was capable of providing size-tunable filtration with a high recovery efficiency (95.25–96.21%) for microbeads with sizes smaller than the defined gap in the filter zone. Furthermore, the proposed device was also capable of performing separation of blood cells and blood plasma from human whole blood. Experimental results showed that an optimum filtration rate of 21.40 and 3.00?μl/min correspond to high recovery efficiencies of 86.69 and 80.66%, respectively, for red blood cells (RBCs) and blood plasma. The separation method developed in this work could be used for various point-of-care diagnostic applications involving separation of plasma and blood cells.     

A multilayer lateral-flow microfluidic device for particle separation

Particle separation technology plays an important role in a wide range of applications as a critical sample preprocessing step for analysis. In this work, we proposed and fabricated a multilayer lateral-flow particle filtration and separation device based on polydimethylsiloxane molding and transfer bonding techniques. Particle separation capability was demonstrated by 4.5-um polystyrene bead filtration and cancer cell (SK-BR-3) retrieving. This device exhibits higher throughput compared with most active particle separation methods and is less vulnerable to membrane clogging problem. This novel multilayer particle filtration and separation device is expected to find applications in biomedical, environmental and microanalysis fields.     

Development of a robust algorithm for detection of nuclei of white blood cells in peripheral blood smear images

Multimedia Tools and Applications - Microscopic evaluation of peripheral blood smear analysis is a commonly used laboratory procedure to diagnose various diseases such as anemia, malaria, leukemia,...     

Binary tree versus single level tree classification of white blood cells

For the problem of white blood cell recognition, the use of various binary tree classification schemes is compared with the application of single tree classifiers.

In principle, in a multi-class problem, binary tree classifiers have the advantage that only a restricted number of features per branch point is needed, enabling an economical design of the classification process, taking into account prior probabilities for all classes.

While these reasons remain valid, the results presented here show that binary tree classifiers do not necessarily improve the correct recognition rate.     

A novel segmentation algorithm for nucleus in white blood cells based on low-rank representation

White blood cells (WBCs) segmentation is a challenging problem in the study of automated morphological systems, due to both the complex nature of the cells and the uncertainty that is present in video microscopy. This paper investigates how to boost the effects of region-based nucleus segmentation in WBCs by means of optimal thresholding and low-rank representation. The main idea is firstly using optimal thresholding to obtain the possible uniform WBC regions in the input image. After that, a manifold-based low-rank representation technique is employed to infer a unified affinity matrix that implicitly encodes the segmentation of the pixels of possible WBC regions. This is achieved by separating the low-rank affinities from the feature matrix into a pair of sparse and low-rank matrices. The experiments show that the proposed method is possible to produce better segmentation results compared with existing approaches.