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.     

Microgravity laser-photophoresis of high density microparticles in water

Laser-photophoresis is a new technique, which can be used to characterize and separate microparticles in liquids. The photophoretic migration of high density solid particles in water has been observed experimentally for the first time by experiments under microgravity conditions. The photophoretic velocity was measured under microgravity conditions, in order to minimize the effects of density difference and convection. Furthermore, by using an optical cylindrical cell, we could observe the precise photophoretic velocities without the wall-induced drag effect. The apparatus consisted of a cw Nd:YAG laser (532 nm), a microscope, a CCD system, and a remote controlled sample stage and was set in a capsule which was used for a free-fall experiment. All the experimental operations were made externally by using a personal computer. The photophoretic velocities for the particles of carbon, stainless steel, gold plated nickel, and polystyrene in water were determined under microgravity. It was found that the photophoretic efficiencies of the photo-absorbing carbon particles and the photo-reflecting metal particles were much larger than those of transparent particles. The order of magnitude of the observed photophoretic efficiency was carbon>stainless steel>gold plated nickel>polystyrene. The photophoretic efficiencies were compared with those calculated by a Mie scattering theory. It was proved that the Mie scattering theory was useful for the prediction of the photophoretic efficiency of various kinds of particles in water.     

Electrokinetic transport of red blood cells in microcapillaries

Electrokinetic flow of a suspension of erythrocytes (red blood cells, RBCs) in 20 num cylindrical fused-silica capillaries is examined in the present work. Flow direction anomalies are observed experimentally and tentatively explained by the development of a pH gradient between the cathode well and the anode well due to electrolysis reactions at the electrodes. This pH gradient alters the local zeta potentials of both the capillary and the RBC and thus the local electroendosmotic liquid flow (EOF) velocities and RBC electrophoretic (EP) velocities. The two velocities are opposite in direction but with EOF dominating such that the RBC moves toward the cathode, opposite to the anode migration observed in bulk conditions. The opposing zeta potentials also lead to RBC aggregation at the anode end for low fields less than 25 V/cm. As the electroendosmotic velocity decreases at the anode end due to decreasing pH, pressure-driven back flow develops to oppose the original EOF at the remaining portions of the capillary ensuring constant fluid flux. When the anode EOF velocity is smaller in magnitude than the EP velocity, reversal of blood cell transport is observed after a short transient time in which a pH gradient forms. RBC velocities and pH dependencies on electric field and MgCl(2) concentration are presented along with data showing the accumulation of charge separation across the capillary. Also, a short-term solution to the pH gradient formation is presented that could help thwart development of pH gradients in micro-devices at lower voltages.     

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.     

Feasibility study of red blood cell debulking by magnetic field-flow fractionation with step-programmed flow

Emerging applications of rare cell separation and analysis, such as separation of mature red blood cells from hematopoietic cell cultures, require efficient methods of red blood cell (RBC) debulking. We have tested the feasibility of magnetic RBC separation as an alternative to centrifugal separation using an approach based on the mechanism of magnetic field-flow fractionation (MgFFF). A specially designed permanent magnet assembly generated a quadrupole field having a maximum field of 1.68 T at the magnet pole tips, zero field at the aperture axis, and a nearly constant radial field gradient of 1.75 T/mm (with a negligible angular component) inside a cylindrical aperture of 1.9 mm (diameter) and 76 mm (length). The cell samples included high-spin hemoglobin RBCs obtained by chemical conversion of hemoglobin to methemoglobin (met RBC) or by exposure to anoxic conditions (deoxy RBC), low-spin hemoglobin obtained by exposure of RBC suspension to ambient air (oxy RBC), and mixtures of deoxy RBC and cells from a KG-1a white blood cell (WBC) line. The observation that met RBCs did not elute from the channel at the lower flow rate of 0.05 mL/min applied for 15 min but quickly eluted at the subsequent higher flow rate of 2.0 mL/min was in agreement with FFF theory. The well-defined experimental conditions (precise field and flow characteristics) and a well-established FFF theory verified by studies with model cell systems provided us with a strong basis for making predictions about potential practical applications of the magnetic RBC separation.

Photophoretic velocimetry--a new way for the in situ determination of particle size distribution and refractive index of hydrocolloids

The migration of particles induced by the forces of light is known as photophoresis. The photophoretic velocity of particles under the influence of a strong light source, e.g. a laser beam, is recorded and measured by means of digital image processing which is called photophoretic velocimetry (PPV). The photophoretic velocity depends on the particle size as well as on its refractive index. By applying PPV, we show the determination of the intrinsic properties of polystyrene (PS), melamine resin (MF) and SiO(2) particles, deduced from velocity distributions of both monodisperse as well as polydisperse suspensions. Especially the continuous determination of the refractive index of single hydrocolloids is of great interest for quality assurance and the discrimination of polydisperse biological or inorganic samples. Also, PPV is not restricted to in water dispersed samples only.     

Impact of Photodynamic Treatment with Meso-substituted Porphyrin on the Immunomodulatory Capacity of White Blood Cell-containing Red Blood Cell Products

After transfusion, the presence of contaminating white blood cells (WBC) in blood components may result in either deleterious or positive immunological responses. We have previously reported that photodynamic treatment (PDT) with meso-substituted mono-phenyl-tri-( N -methyl-4-pyridyl)-porphyrin (Tri-P(4)) and red light can inactivate pathogens in red blood cell (RBC) products. The present study explored the effect of PDT on contaminating WBC in RBC products with varying hematocrit (Hct). After PDT, we evaluated adaptive and innate immunomodulation through allogeneic and mitogenic stimulation. PDT resulted in decreased T-cell proliferation which was more pronounced with lower Hct. Dark effect of porphyrin Tri-P(4) was remarkable on antigen-presenting cells affecting expression of co-stimulatory molecules CD80/CD86. Finally, cytokine profile after PDT revealed a mixed Th1/Th2 type response while surface antigen expression supported the development of alternatively activated macrophages (AAMφ or Type 2 macrophages) instead of dendritic cells. In conclusion, PDT with Tri-P(4) altered proliferation, allo-stimulation, cell surface antigen expression and cytokine profiles of the cells. These results suggest that PDT may be potentially useful in preventing transfusion-associated graft- versus -host disease and alloimmunization. It seems worthwhile to further explore PDT-induced immunomodulation to optimize conditions which may result in allo-tolerance by AAMφ.     

Characteristics and function of human hemoglobin vesicles as an oxygen carrier

A liposome‐encapsulated human hemoglobin (Neo Red Cell, (NRC) has been developed and evaluated as an artificial oxygen carrier. The NRC is a liposome‐encapsulated highly concentrated (>45%) stroma‐free human hemoglobin with inositol hexaphosphate (IHP as an allosteric effector), a coenzyme and substrates for reducing methemoglobin (metHb). The NRC's surface was coated with polyethylene glycol to prevent aggregation in plasma and to prolong their retention time in the blood stream. The oxygen binding behavior of the NRC in vitro was investigated and it was found that it effectively transports oxygen in vivo as an oxygen carrier. The oxygen binding behavior and kinetics were studied by the stopped‐flow method and the oxygen binding curve of the NRC was determined. The oxygen binding speed and binding coefficient (K_on) of NRC, washed human red blood cells (WRBC) and stroma‐free human hemoglobin (SFHb) were measured by stopped‐flow method. The oxygen binding speed of SFHb was the highest, while that of RBC was the lowest and that of NRC was intermediate. The oxygen binding of NRC ended within 60 msec when deoxy‐NRC was mixed with oxygen. The K_on of NRC was 2.9 × 10⁵, 10 times faster than that of RBC. The oxygen binding curve and P₅₀O₂ of NRC that contained various IHP concentrations were measured. The oxygen‐binding curve of the NRC sequentially shifted to the right as the IHP content was increased. Exchange transfusion of 70% was carried out for rats with NRC containing various concentrations of IHP and of Hb, and investigated the optimum concentration of NRC in vivo. The lactate value after exchange transfusion was three times higher than before exchange transfusion, when rats were subjected to exchange transfused with NRC that did not contain IHP. But the increase of lactate was suppressed when rats were transfused with NRC that contained IHP. When the Hb concentration of NRC was 5 and 6%, exchange transfused rats recovered to normality just like rats transfused with RBC. Copyright © 2000 John Wiley & Sons, Ltd.     

The voltammetric behavior of bone marrow of leukaemia and its clinical application

The electrochemical voltammetric behavior of bone marrow of leukaemia has been investigated by a self-devised cytosensing system. The two anodic peaks of erythrocytes (red blood cells, RBC) in bone marrow of leukaemia appeared at 0.73 +/- 0.03 and 0.83 +/- 0.02V vs. SCE, respectively, on the first scan. The anodic peak of leukocytes (white blood cells, WBC) appeared at 0.32 +/- 0.03V vs. SCE. The anodic peak of RBC at 0.83V disappeared when the patients were cured. The experimental results show that the voltammetric behavior of erythrocytes is in constant contact with the initial stage of leukaemia. The cyclic voltammetric behavior of 40 cases of leukaemia including acute myeloid leukaemia (AML) and chronic myeloid leukaemia (CML) and 10 cases of healthy volunteer peripheral blood was studied. The cyclic voltammetric behavior of erythrocytes may provide a simple and specific marker for diagnosis of leukaemia.     

Functional evaluation of hemoglobin-and lipidheme-vesicles as red cell substitutes

The two kinds of red cell substitutes, hemoglobin-vesicles (HbV) and lipidheme-vesicles (LihV, totally synthetic oxygen carrier), were evaluated in terms of physicochemical properties such as binding and dissociating reactions of ligands (CO, O₂ and NO), rheological and structural properties. Carbonylation of Hb during the purification of Hb and the preparation of HbV is effective to prevent Hb denaturation. The rates of oxygenation of both HbV and LihV are faster than that of red blood cells (RBC). Their oxygen affinities (P₅₀, HbV, 32 mmHg; LihV, 43 mmHg, cf. RBC, 28 mmHg) can be controlled to transport a sufficient amount of oxygen comparable with that of RBC. The smaller sizes of vesicles are advantageous for prompt ligand reaction and low viscosity. Both HbV and RBC show about 100 times less vasoconstrictive effects than stripped Hb. HbV shows only one sixth of the slow binding rate of NO (=endothelial derived relaxation factor) in comparison with stripped Hb. Inhibition of vasoconstriction by those vesicles is discussed from the kinetic data.     

Penetration of laser light through red blood cell ghosts

Hemoglobin is the main absorber of visible light in blood and blood-perfused tissues. However, hemoglobin is released from a red blood cell (RBC) during hemolysis. Hemolysis may be caused by a large number of medical conditions, including photodynamic therapy (PDT) and this subsequently can affect passage of light through the treated biological structures. The purpose of the present study was to determine the penetration of a laser beam through a suspension of hemoglobin-free human red blood cells (RBCs) - ghosts. Although hemoglobin has been efficiently removed from the samples used in our experiments, our measurements show that the samples still effectively attenuate the radiant power of penetrating laser light. We established penetration depths of 12.6mm and 15.4mm for two different laser light wavelengths, 532nm and 630nm, respectively. The penetration depth of laser light was about one order of magnitude higher for hemoglobin-free RBC ghosts as compared to intact RBCs [8,10,12]. These results can be important in case of phototherapy or biostimulation, since all photons that penetrate in a biological object may interact with it and evoke biological response.     

Photophoretic assembly and migration of dielectric particles and Escherichia coli in liquids using a subwavelength diameter optical fiber

We demonstrate a photophoretic assembly and migration of dielectric (SiO(2) and TiO(2)) particles and bacteria (Escherichia coli) in liquids by using a subwavelength diameter fiber. With a lightwave at 1.55 μm launched into the fiber, the objects are radiated by the leaking light of the fiber to yield negative photophoretic forces which drive the objects to move toward the fiber, with an average assembling/migrating speed of 5-15 individuals per second (ind/s). The influences of laser-on duration, optical power, and size of particles on the photophoretic velocities are also investigated.     

In vitro evaluation of cellulose acetate hemodialyzer immobilized with heparin

In this study, heparin was immobilized onto cellulose acetate hollow fibers to improve the anticoagulation performance during hemodialysis. In vitro evaluation was carried out using mini‐hemodialyzer circulating with fresh porcine whole blood to simulate kidney therapy. The dialysis performance and hemocompatibility were estimated. The results showed that heparinized hemodialyzer could be used through out the whole dialysis time (4 hr) without injecting additional heparin to prevent coagulation in the dialysis system. In addition, the hemocompatibility was evaluated by measuring activated partial thromboplastin time (APTT), prothrombin time (PT), and fibrinogen time (FT). The complete blood count (CBC) including red blood cell (RBC), hemoglobin (Hgb), hematocrit (Hct), white blood cell (WBC), and platelet were determined. The results showed that heparinization could keep the CBC stable during dialysis, whereas unmodified cellulose acetate hemodialyzer would cause a decrease in RBC unless heparin was injected during dialysis. Heparinized hemodialyzer showed longer APTT, PT, and FT than unmodified hemodialyzer. Heparinized hemodialyzer also showed slightly higher clearance than unmodified hemodialyzer. These results indicated that the dialysis performance and hemocompatibility of cellulose acetate hemodialyzer could be improved by the immobilization of heparin. Copyright © 2006 John Wiley & Sons, Ltd.     

Microbe removal using a micrometre-sized optical fiber

The growing global shortage of fresh water has lead to the need for technological innovations for water purification and reuse. The removal of pathogenic microbes from urban, laboratory or industrial wastewater is one of the most challenging and critical issues due to the potential risk of microbe outbreaks. In addition, microbe removal in human blood or tissues has also inspired novel techniques for extracting and collecting different cells in fluidic channels or vessels. Recently, efficient removal of microbes from flowing water running under gravity feed has been achieved using filters in nanotubes or nanofibers. Here we report a highly efficient removal of microbes from flowing water in a fluidic channel using a reusable micrometre-sized optical fiber. Our technique is based on photophoresis of the microbes induced by the radiation of 1.55 μm wavelength injected into the fiber. Yeast cell suspensions, as a sample of microbe-contaminated water, are flown through a fluidic channel and the suspended cells are collected by the photophoretic forces, leading to a consistent accumulation of the yeast cells. The experiments indicate that a removal efficiency of 99.9% can be obtained when the flow velocity of the suspensions is less than the peak photophoretic velocity of the yeast cells.     

Photodynamic Treatment of Red Blood Cell Concentrates For Virus Inactivation Enhances Red Blood Cell Aggregation: Protection with Antioxidants

Abstract— Photodynamic treatment (PDT) using phthalocyanines and red light appears to be a promising procedure for decontamination of red blood cell (RBC) concentrates for transfusion. A possible complication of this treatment may be induced aggregation of RBC. The production of RBC aggregates was measured with a novel computerized cell flow properties analyzer (CFA). The PDT of RBC concentrates with sulfonated aluminum phthalocy-anine (AIPcS4) and the silicon phthalocyanine Pc 4 under virucidal conditions markedly enhanced RBC aggregation and higher shear stress was required to disperse these aggregates. The clusters of cells were huge and abnormally shaped, unlike the rouleaux formed by untreated RBC. This aggregation was prevented when a mixture of antioxidants was included during PDT. Addition of the antioxidants after PDT reduced aggregation only partially. It is concluded that inclusion of antioxidants during PDT of RBC concentrates prior to transfusion may reduce or eliminate the hemodynamic risk that the virucidal treatment may present to the recipient.     

Determination of erythrocyte deformability and its correlation to cellular ATP release using microbore tubing with diameters that approximate resistance vessels in vivo

A novel method is described for measuring the deformability of red blood cells (RBCs) in tubing whose diameters approximate forces encountered in vivo. Here, RBCs from rabbits are loaded into a 50 cm section of 75 microm id microbore tubing and connected to a syringe pump. This section of tubing is then connected to a 15 cm section of 25 microm id tubing. As buffer is pumped through the flow system, the RBCs are evacuated from both sections of tubing. However, the inability of the RBCs to move freely through the 25 mirom id section of tubing results in a buildup of cells at the inlet of this portion of tubing. The continued force output by the syringe pump results in a deformation of the RBCs until all of the cells are eventually evacuated from the flow system. It was found that a measurement of the time required to reach half of the maximum pressure (1/2 P(max)) may be used as an indicator of the RBC deformability. For a given sample, a simple buffer results in less time to reach 1/2 P(max) (6.9 +/- 0.2 s) than deformable RBCs (21.6 +/- 0.8 s). To verify that the increased amount of time to reach 1/2 P(max) is indeed due to the RBCs, various hematocrits of an RBC sample were investigated and, as expected, it was found that a 12% RBC hematocrit had a higher 1/2 P(max) value (26.0 s +/- 2.2 s) when compared to a 7% hematocrit (19.1 +/- 0.3 s). In addition, RBCs chemically stiffened with glutaraldehyde were shown to be 25% less deformable than normal RBCs. Finally, a study was performed to examine the relationship between RBC deformability and ATP release and it was found that ATP release increased as a function of RBC deformability. This method greatly simplifies deformability measurements, employing only a syringe pump and microbore tubing, and may lead to a more complete understanding of the physiological significance of erythrocyte deformability.     

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.     

Kinetic study of all-or-none hemolysis induced by cationic amphiphilic polymethacrylates with antimicrobial activity

To gain an understanding of the toxicity of antimicrobial polymers to human cells, their hemolytic action was investigated using human red blood cells (RBCs). We examined the hemolysis induced by cationic amphiphilicmethacrylate random copolymers, which have amino ethyl sidechains as cationic units and either butyl or methyl methacrylate as hydrophobic units. The polymer with 30 mol% butyl sidechains (B30) displayed higher hemolytic toxicity than the polymer with 59 mol% methyl sidechains (M59). B30 also induced faster release of hemoglobin from RBCs than M59. A new theoretical model is proposed based on two consecutive steps to form active polymer species on the RBC membranes, which are associated to RBC lysis. This model takes the all-or-none release of hemoglobin by the rupture of RBCs into account, providing new insight into the polymer-induced hemolysis regarding how individual or collective cells respond to the polymers.     

2.45 GHz Microwave Irradiation-Induced Oxidative Stress Affects Implantation or Pregnancy in Mice, Mus musculus

The present experiment was designed to study the 2.45 GHz low-level microwave (MW) irradiation-induced stress response and its effect on implantation or pregnancy in female mice. Twelve-week-old mice were exposed to MW radiation (continuous wave for 2 h/day for 45 days, frequency 2.45 GHz, power density?=?0.033549 mW/cm², and specific absorption rate?=?0.023023 W/kg). At the end of a total of 45 days of exposure, mice were sacrificed, implantation sites were monitored, blood was processed to study stress parameters (hemoglobin, RBC and WBC count, and neutrophil/lymphocyte (N/L) ratio), the brain was processed for comet assay, and plasma was used for nitric oxide (NO), progesterone and estradiol estimation. Reactive oxygen species (ROS) and the activities of ROS-scavenging enzymes— superoxide dismutase, catalase, and glutathione peroxidase—were determined in the liver, kidney and ovary. We observed that implantation sites were affected significantly in MW-irradiated mice as compared to control. Further, in addition to a significant increase in ROS, hemoglobin (p?<?0.001), RBC and WBC counts (p?<?0.001), N/L ratio (p?<?0.01), DNA damage (p?<?0.001) in brain cells, and plasma estradiol concentration (p?<?0.05), a significant decrease was observed in NO level (p?<?0.05) and antioxidant enzyme activities of MW-exposed mice. Our findings led us to conclude that a low level of MW irradiation-induced oxidative stress not only suppresses implantation, but it may also lead to deformity of the embryo in case pregnancy continues. We also suggest that MW radiation-induced oxidative stress by increasing ROS production in the body may lead to DNA strand breakage in the brain cells and implantation failure/resorption or abnormal pregnancy in mice.     

Laser photophoretic migration with periodic expansion-contraction motion of photo-absorbing microemulsion droplets in water

When the water-in-oil (w/o) microemulsion droplets including the Co(III)-pyridylazo complex as the photo-absorber were irradiated with a continuous-wave Ar(+) ion laser (514.5 nm), we have observed unexpected phenomenon that photo-absorbing microemulsion droplets in water repeated the expansion and the sudden contraction during the laser photophoretic migration. The period of the expansion-contraction cycle was inversely proportional to both the concentration of the complex and the irradiated laser power and was independent of the initial size of the droplet. The mechanism of the periodic motion of the droplet was investigated by local temperature measurement and Raman microscope spectroscopy. It was suggested that the first step was the phase separation of the bicontinuous microemulsion droplet into the normal w/o microemulsion outer phase and the aqueous inner phase in the droplet, which was caused by the laser-induced temperature gradient inside the droplet. Subsequently, an expansion of the inner aqueous phase was induced by the percolation of the external water by thermo-osmosis, which was caused by the laser-induced temperature gradient between the inside and the outside of the microemulsion liquid membrane of the droplet. When the liquid membrane became thinner to a critical thickness, the inner aqueous phase was released and the droplet shrank into the original size. The proposed mechanism can give an account of the unique cyclical motion.