Capillary penetration failure of blood suspensions

Blood suspension fails to penetrate a capillary with radius R less than 50 microm even if the capillary is perfectly wettable. This invasion threshold is attributed to three red blood cells (RBCs) segregation mechanisms--corner deflection at the entrance, the intermediate deformation-induced radial migration and shear-induced diffusion within a packed slug at the meniscus. The shear-induced radial migration for deformable particles endows the blood cells with a higher velocity than the meniscus to form the concentrated slug behind the meniscus. This tightly packed slug has a higher resistance and arrests the flow. Rigid particles and rigidified blood cells result in wetting behavior similar to that seen for homogeneous liquids, with decreased RBC migration towards the capillary centerline and reduction of packing. Corner deflection with a radial drift velocity accelerates the radial migration for small capillaries. However, deformation-induced radial migration is the key mechanism responsible for penetration failure. This sequence of mechanisms is confirmed through videomicroscopy and scaling theories were applied to capture the dependence of the critical capillary radius as a function of RBC concentrations.     

Chemiluminescence detection of ATP release from red blood cells upon passage through microbore tubing

A novel approach for the determination of adenosine triphosphate (ATP) released from red blood cells (RBCs) after passage through microbore capillaries is described. ATP is often released from RBCs in vessels and has been linked to the production of nitric oxide, a known vasodilator. The system described here uses a syringe pump to deliver microliter flow rates (5-15 microl min(-1)) of reagent and sample through fused silica capillary tubing of varying dimensions (25-75 microm) to a photomultiplier tube. The released ATP is characterized by the detection of chemiluminescent emission from the luciferin-luciferase reaction. The amount of ATP released is directly proportional to the number of RBCs injected into the system. Results also suggest that the amount of released ATP decreases from 6.9 microM to 1.4 microM as the tubing diameter is increased from 25 microm to 75 microm. An investigation of capillary lengths ranging from 15 to 35 cm resulted in ATP concentrations of 1.5 microM to 2.4 microM being released. Results also indicate that increases in flow rate also induce increased amounts of ATP release. These results are consistent with those of previous systems attempting to model the physiological release of ATP from red blood cells.     

Direct measurement of the impact of impaired erythrocyte deformability on microvascular network perfusion in a microfluidic device

The ability of red blood cells (RBCs, erythrocytes) to deform and pass through capillaries is essential for continual flow of blood in the microvasculature, which ensures an adequate supply of oxygen and nutrients, prompt removal of metabolic waste products, transport of drugs and hormones, and traffic of circulating cells to and from all living tissues. This paper presents a novel tool for evaluating the impact of impaired deformability of RBCs on the flow of blood in the microvasculature by directly measuring perfusion of a test microchannel network with dimensions and topology similar to the real microcirculation. The measurement of microchannel network perfusion is compared with RBC filtration -- a conventional assay of RBC deformability. In contrast to RBC filterability, network perfusion depends linearly on RBC deformability modulated by graded exposure to glutaraldehyde, showing a higher sensitivity to small changes of deformability. The direct measurement of microchannel network perfusion represents a new concept for the field of blood rheology and should prove beneficial for basic science and clinical applications.     

A microfabricated deformability-based flow cytometer with application to malaria

Malaria resulting from Plasmodium falciparum infection is a major cause of human suffering and mortality. Red blood cell (RBC) deformability plays a major role in the pathogenesis of malaria. Here we introduce an automated microfabricated "deformability cytometer" that measures dynamic mechanical responses of 10(3) to 10(4) individual RBCs in a cell population. Fluorescence measurements of each RBC are simultaneously acquired, resulting in a population-based correlation between biochemical properties, such as cell surface markers, and dynamic mechanical deformability. This device is especially applicable to heterogeneous cell populations. We demonstrate its ability to mechanically characterize a small number of P. falciparum-infected (ring stage) RBCs in a large population of uninfected RBCs. Furthermore, we are able to infer quantitative mechanical properties of individual RBCs from the observed dynamic behavior through a dissipative particle dynamics (DPD) model. These methods collectively provide a systematic approach to characterize the biomechanical properties of cells in a high-throughput manner.     

An inexpensive thread-based system for simple and rapid blood grouping

This study investigates the use of thread as a flexible and low-cost substrate for the rapid grouping of blood. The use of a capillary substrate such as thread for blood grouping utilises the sensitivity of the flow resistance of large particles in narrow capillary channels to separate agglutinated red blood cells (RBCs) from plasma. Large and discrete particles formed in a continuous liquid phase do not provide capillary wicking driving force and fall behind the capillary wicking front, leading to their separation from the wicking liquid. The capillary substrate therefore provides a very promising but different mechanism for the separation of the agglutinated RBCs and the blood serum phase compared to most existing blood grouping methods. The principle of chromatographic separation is also exploited in this study via the use of suitable dyes to enhance the visual detection of the agglutinated RBCs and the serum phase; surprising and encouraging outcomes are obtained. Using a thread-based device, the ABO and Rh groups can be successfully determined with only 2 μL of whole blood from a pricked finger tip within 1 min and without pre-treatment of the blood sample. It is hoped that a new, inexpensive, rapid and simple method may provide an easy-to-use blood grouping platform well suited to those in developing or remote regions of the world.     

Traffic of leukocytes in microfluidic channels with rectangular and rounded cross-sections

Traffic of leukocytes in microvascular networks (particularly through arteriolar bifurcations and venular convergences) affects the dynamics of capillary blood flow, initiation of leukocyte adhesion during inflammation, and localization and development of atherosclerotic plaques in vivo. Recently, a growing research effort has been focused on fabricating microvascular networks comprising artificial vessels with more realistic, rounded cross-sections. This paper investigated the impact of the cross-sectional geometry of microchannels on the traffic of leukocytes flowing with human whole blood through a non-symmetrical bifurcation that consisted of a 50 μm mother channel bifurcating into 30 μm and 50 μm daughter branches. Two versions of the same bifurcation comprising microchannels with rectangular and rounded cross-sections were fabricated using conventional multi-layer photolithography to produce rectangular microchannles that were then rounded in situ using a recently developed method of liquid PDMS/air bubble injection. For microchannels with rounded cross-sections, about two-thirds of marginated leukocytes traveling along a path in the top plane of the bifurcation entered the smallest 30 μm daughter branch. This distribution was reversed in microchannels with rectangular cross-sections--the majority of leukocytes traveling along a similar path continued to follow the 50 μm microchannels after the bifurcation. This dramatic difference in the distribution of leukocyte traffic among the branches of the bifurcation can be explained by preferential margination of leukocytes towards the corners of the 50 μm mother microchannels with rectangular cross-sections, and by the additional hindrance to leukocyte entry created by the sharp transition from the 50 μm mother microchannel to the 30 μm daughter branch at the intersection. The results of this study suggest that the trajectories of marginated leukocytes passing through non-symmetrical bifurcations are significantly affected by the cross-sectional geometry of microchannels and emphasize the importance of using microfludic systems with geometrical configurations closely matching physiological configurations when modeling the dynamics of whole blood flow in the microcirculation.     

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

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

A discrete-particle model of blood dynamics in capillary vessels

We investigate the mechanism of aggregation of red blood cells (RBC) in capillary vessels. We use a discrete-particle model in 3D to model the flow of plasma and RBCs within a capillary tube. This model can accurately capture the scales from 0.001 to 100 microm, far below the scales that can be modeled numerically with classical computational fluid dynamics. The flexible viscoelastic red blood cells and the walls of the elastic vessel are made up of solid particles held together by elastic harmonic forces. The plasma is represented by a system of dissipative fluid particles. Modeling has been carried out using 1 to 3 million solid and fluid particles. We have modeled the flow of cells with vastly different shapes, such as normal and "sickle" cells. The two situations involving a straight capillary and a pipe with a choking point have been considered. The cells can coagulate in spite of the absence of adhesive forces in the model. We conclude that aggregation of red blood cells in capillary vessels can be stimulated by depletion forces and hydrodynamic interactions. The cluster of "sickle" cells formed in the choking point of the capillary efficiently decelerates the flow, while normal cells can pass through. These qualitative results from our first numerical results accord well with the laboratory findings.     

Preparation of red blood cell column for capillary electrochromatography.

A column packed with red blood cells (RBCs) was prepared for electrochromatography as a separation and reaction column. RBCs were kept inside a piece of fused silica capillary tubing with 2% agarose gel. In the column, RBCs were uniformly distributed in the agarose gel matrix and their electrophoretic movements due to an applied voltage were suppressed well. The durability of the biological function of the column under applied voltage was about 1 h, although it could remain for 2-3 days without applied voltage. The column could not be used when hemolysis of the RBCs was observed in the column. When the developed "RBC-gel column" was used, both pyridoxamine and serotonin were converted to other compounds through their direct contact with RBCs.     

Sequencing of real-world samples using a microfabricated hybrid device having unconstrained straight separation channels

We describe a microfabricated hybrid device that consists of a microfabricated chip containing multiple twin-T injectors attached to an array of capillaries that serve as the separation channels. A new fabrication process was employed to create two differently sized round channels in a chip. Twin-T injectors were formed by the smaller round channels that match the bore of the separation capillaries and separation capillaries were incorporated to the injectors through the larger round channels that match the outer diameter of the capillaries. This allows for a minimum dead volume and provides a robust chip/capillary interface. This hybrid design takes full advantage, such as sample stacking and purification and uniform signal intensity profile, of the unique chip injection scheme for DNA sequencing while employing long straight capillaries for the separations. In essence, the separation channel length is optimized for both speed and resolution since it is unconstrained by chip size. To demonstrate the reliability and practicality of this hybrid device, we sequenced over 1000 real-world samples from Human Chromosome 5 and Ciona intestinalis, prepared at Joint Genome Institute. We achieved average Phred20 read of 675 bases in about 70 min with a success rate of 91%. For the similar type of samples on MegaBACE 1000, the average Phred20 read is about 550-600 bases in 120 min separation time with a success rate of about 80-90%.     

DNA sequencing by capillary array electrophoresis and microfabricated array systems

To comply with the current needs for high-speed DNA sequencing analysis, several instruments and innovative technologies have been introduced by several groups in recent years. This review article discusses and compares the issues regarding high-throughput DNA sequencing by electrophoretic methods in miniaturized systems, such as capillaries, capillary arrays, and microchannels. Initially, general features of several capillary array designs (including commercial ones) will be considered, followed by similar analyses with microfabricated array electrophoretic devices and how they can contribute to the success of large sequencing projects.     

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

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

Non-adsorbing macromolecules in plasma induce erythrocyte adhesion to the endothelium

Red blood cell (RBC) adhesion to the endothelium is usually insignificant. However, an enhanced adhesion can be observed in various pathological conditions such as diabetes mellitus or sickle cell disease, which is often accompanied by elevated levels of pro-adhesive plasma proteins such as fibrinogen. In the past, these proteins have only been considered to act as ligands, cross-linking the corresponding receptors on adjacent cells, but the detailed underlying mechanism often remained obscure. This work demonstrates that the presence of non-adsorbing polymers in plasma can also enhance the adhesion efficiency of RBCs to endothelial cells (ECs) through depletion interaction. Furthermore, adhesion of RBCs to ECs may be likewise promoted by the protein fibrinogen through depletion interaction. We propose an alternative mechanism for the pro-adhesive effects of plasma proteins and indicate that depletion interaction might play a significant role for the stabilization and destabilization of blood flow in health and disease.     

活体小鼠中单个红细胞的拉曼光谱分析

应用拉曼光谱与光镊技术对活体小鼠毛细动脉和静脉血管中的全血以及毛细血管中的红细胞和体外的全血及单个红细胞进行研究;光谱分析显示,可以获得活体血液的拉曼光谱;在动脉毛细管和静脉毛细管中可以观察到清晰的携氧与去氧血液的不同光谱;体内血管中血红细胞的血红蛋白浓度比体外的更高,在动脉毛细管中的血红细胞的携氧态特征峰明显,且具有...     

Estimation of electrophoretic mobilities of red blood cells in 1-G and microgravity using a miniature capillary electrophoresis unit

Electrophoretic mobilities of red blood cells (RBCs) were measured in microgravity using a home-made capillary electrophoresis unit, which consisted of two small reservoirs of 0.6 mL and a fused-silica capillary tubing with 2 cm in length and 50 num in inner diameter. Migration of RBCs was observed by a microscope at 1000 times magnification and recorded on a videotape. The experiments were performed during stays in microgravity (about 0.01 G), which lasted 20 s and were attained by parabolic flights of an aircraft. On average, the electrophoretic mobilities of RBCs determined in microgravity were about 30% higher than those measured at 1-G condition irrespectively whether the cells were suspended in saline or serum during measurements. This difference might be explained as being mainly due to the cell floating in microgravity. Morphological changes of RBCs may contribute partly to the difference, while the variation in viscosity of the medium under microgravity could play only a minor role.     

Automated sampling with capillaries

A system is described for the analysis of serum contained in capillaries. The capillaries, filled with samples, are placed directly into a moving stream of diluent which flushes the capillaries, carrying the samples into a continuous flow or discrete system of analysis. The capillaries are inserted into holes in a plastic block which is pushed forward sequentially by a drive mechanism. As each capillary comes into line with an entrance tube and exit tube, reagent is pumped through these tubes and through the capillary. As an alternative, a dispenser is attached to the inlet tube, and as each capillary comes into position, a measured amount of liquid is dispensed through the capillary and into a container. The system is applied to continuous flow analysis of phosphate, alkaline phosphatase, uric acid, and creatinine. The construction of an efficient and reliable peristaltic pump is described for the continuous flow system.     

A microfabricated electrical differential counter for the selective enumeration of CD4+ T lymphocytes

We have developed a microfabricated biochip that enumerates CD4+ T lymphocytes from leukocyte populations obtained from human blood samples using electrical impedance sensing and immunoaffinity chromatography. T cell counts are found by obtaining the difference between the number of leukocytes before and after depleting CD4+ T cells with immobilized antibodies in a capture chamber. This differential counting technique has been validated to analyze physiological concentrations of leukocytes with an accuracy of ～9 cells per μL by passivating the capture chamber with bovine serum albumin. In addition, the counter provided T cell counts which correlated closely with an optical control (R(2) = 0.997) for CD4 cell concentrations ranging from approximately 100 to 700 cells per μL. We believe that this approach can be a promising method for bringing quantitative HIV/AIDS diagnostics to resource-poor regions in the world.     

High-throughput biophysical measurement of human red blood cells

This paper reports a microfluidic system for biophysical characterization of red blood cells (RBCs) at a speed of 100-150 cells s(-1). Electrical impedance measurement is made when single RBCs flow through a constriction channel that is marginally smaller than RBCs' diameters. The multiple parameters quantified as mechanical and electrical signatures of each RBC include transit time, impedance amplitude ratio, and impedance phase increase. Histograms, compiled from 84,073 adult RBCs (from 5 adult blood samples) and 82,253 neonatal RBCs (from 5 newborn blood samples), reveal different biophysical properties across samples and between the adult and neonatal RBC populations. In comparison with previously reported microfluidic devices for single RBC biophysical measurement, this system has a higher throughput, higher signal to noise ratio, and the capability of performing multi-parameter measurements.     

Microfabricated liquid junction hybrid capillary electrophoresis‐mass spectrometry interface for fully automated operation

One of the challenging instrumental aspects in coupling an automated CE instrument with ESI mass spectrometry (CE‐MS) is finding the balance between the stability, reproducibility and sensitivity of the analysis and compatibility with the standard CE instrumentation. Here, we present a development of a new liquid junction based electrospray interface for automated CE‐MS, with a focus on the technical design followed by computer modeling of transport conditions as well as characterization of basic performance of the interface. This hybrid arrangement designed as a microfabricated unit attachable to the automated CE instrument allows using of a wide range of separation capillaries with respect to their diameter, length or internal coating (e.g., for suppressed electroosmotic flow). Different compositions of the ESI liquid and background electrolyte solutions can be used if needed. The microfabricated part, prepared by laser machining from polyimide, includes a self‐aligning liquid junction, a short transport channel, and a pointed sprayer for the electrospray ionization. This microfabricated part is positioned in a plastic connection block securing the separation capillary and flushing ports. Transport conditions were modelled using computer simulation and the real life performance of the interface was compared to that of a commercial sheath liquid interface. The basic performance of the interface was demonstrated by separations of peptides, proteins, and oligosaccharides.     

Towards stationary phases for chromatography on a microchip: molded porous polymer monoliths prepared in capillaries by photoinitiated in situ polymerization as separation media for electrochromatography

Photoinitiated free radical polymerization has been used for the preparation of porous polymer monoliths within UV transparent fused silica capillaries and quartz tubes. These formats were used as models for the preparation of the separation media within channels of microfabricated devices. A mixture of ethylene dimethacrylate, butyl methacrylate, and 2-acrylamido-2-methyl-1-propanesulfonic acid was polymerized in the presence of a porogenic solvent consisting of 1-propanol, 1,4-butanediol, and water at room temperature under UV irradiation. Modification of the porogen composition enables the tailoring of pore size within the broad range from ca. 100 to 4000 nm. Scanning electron micrographs confirmed the homogeneity of the porous structure of the materials prepared, even in a quartz tube with a diameter as large as 4 mm. Separation properties of the resulting capillary columns were tested in capillary electrochromatography (CEC) mode using a mixture of thiourea and eight aromatic compounds. Plate number as high as 210 000 plates/m were found for a capillary column with optimized porous properties. The monolithic columns were also able to separate mixtures of peptides.