Coalescing drops in microfluidic parking networks: A multifunctional platform for drop-based microfluidics

Multiwell plate and pipette systems have revolutionized modern biological analysis; however, they have disadvantages because testing in the submicroliter range is challenging, and increasing the number of samples is expensive. We propose a new microfluidic methodology that delivers the functionality of multiwell plates and pipettes at the nanoliter scale by utilizing drop coalescence and confinement-guided breakup in microfluidic parking networks (MPNs). Highly monodisperse arrays of drops obtained using a hydrodynamic self-rectification process are parked at prescribed locations in the device, and our method allows subsequent drop manipulations such as fine-gradation dilutions, reactant addition, and fluid replacement while retaining microparticles contained in the sample. Our devices operate in a quasistatic regime where drop shapes are determined primarily by the channel geometry. Thus, the behavior of parked drops is insensitive to flow conditions. This insensitivity enables highly parallelized manipulation of drop arrays of different composition, without a need for fine-tuning the flow conditions and other system parameters. We also find that drop coalescence can be switched off above a critical capillary number, enabling individual addressability of drops in complex MPNs. The platform demonstrated here is a promising candidate for conducting multistep biological assays in a highly multiplexed manner, using thousands of submicroliter samples.     

Simulation of malaria-infected red blood cells in microfluidic channels: Passage and blockage

Malaria-infected red blood cells (iRBCs) become less deformable with the progression of infection and tend to occlude microcapillaries. This process has been investigated in vitro using microfluidic channels. The objective of this paper is to provide a quantitative basis for interpreting the experimental observations of iRBC occlusion of microfluidic channels. Using a particle-based model for the iRBC, we simulate the traverse of iRBCs through a converging microfluidic channel and explore the progressive loss of cell deformability due to three factors: the stiffening of the membrane, the reduction of the cell''s surface-volume ratio, and the growing solid parasites inside the cell. When examined individually, each factor tends to hinder the passage of the iRBC and lengthen the transit time. Moreover, at sufficient magnitude, each may lead to obstruction of narrow microfluidic channels. We then integrate the three factors into a series of simulations that mimic the development of malaria infection through the ring, trophozoite, and schizont stages. These simulations successfully reproduce the experimental observation that with progression of infection, the iRBC transitions from passage to blockage in larger and larger channels. The numerical results suggest a scheme for quantifying iRBC rigidification through microfluidic measurements of the critical pressure required for passage.     

The mechanical properties of stored red blood cells measured by a convenient microfluidic approach combining with mathematic model

The mechanical properties of red blood cells (RBCs) are critical to the rheological and hemodynamic behavior of blood. Although measurements of the mechanical properties of RBCs have been studied for many years, the existing methods, such as ektacytometry, micropipette aspiration, and microfluidic approaches, still have limitations. Mechanical changes to RBCs during storage play an important role in transfusions, and so need to be evaluated pre-transfusion, which demands a convenient and rapid detection method. We present a microfluidic approach that focuses on the mechanical properties of single cell under physiological shear flow and does not require any high-end equipment, like a high-speed camera. Using this method, the images of stretched RBCs under physical shear can be obtained. The subsequent analysis, combined with mathematic models, gives the deformability distribution, the morphology distribution, the normalized curvature, and the Young''s modulus (E) of the stored RBCs. The deformability index and the morphology distribution show that the deformability of RBCs decreases significantly with storage time. The normalized curvature, which is defined as the curvature of the cell tail during stretching in flow, suggests that the surface charge of the stored RBCs decreases significantly. According to the mathematic model, which derives from the relation between shear stress and the adherent cells'' extension ratio, the Young''s moduli of the stored RBCs are also calculated and show significant increase with storage. Therefore, the present method is capable of representing the mechanical properties and can distinguish the mechanical changes of the RBCs during storage. The advantages of this method are the small sample needed, high-throughput, and easy-use, which make it promising for the quality monitoring of RBCs.     

Sorting of circulating tumor cells (MV3-melanoma) and red blood cells using non-inertial lift

We demonstrate the method of non-inertial lift induced cell sorting (NILICS), a continuous, passive, and label-free cell sorting approach in a simple single layer microfluidic device at low Reynolds number flow conditions. In the experiments, we exploit the non-inertial lift effect to sort circulating MV3-melanoma cells from red blood cell suspensions at different hematocrits as high as 9%. We analyze the separation process and the influence of hematocrit and volume flow rates. We achieve sorting efficiencies for MV3-cells up to E_MV3 = 100% at Hct = 9% and demonstrate cell viability by recultivation of the sorted cells.     

Characterizing the dielectric properties of human mesenchymal stem cells and the effects of charged elastin-like polypeptide copolymer treatment

Human mesenchymal stem cells (hMSCs) have three key properties that make them desirable for stem cell therapeutics: differentiation capacity, trophic activity, and ability to self-renew. However, current separation techniques are inefficient, time consuming, expensive, and, in some cases, alter hMSCs cellular function and viability. Dielectrophoresis (DEP) is a technique that uses alternating current electric fields to spatially separate biological cells based on the dielectric properties of their membrane and cytoplasm. This work implements the first steps toward the development of a continuous cell sorting microfluidic device by characterizing native hMSCs dielectric signatures and comparing them to hMSCs morphologically standardized with a polymer. A quadrapole Ti-Au electrode microdevice was used to observe hMSC DEP behaviors, and quantify frequency spectra and cross-over frequency of hMSCs from 0.010–35 MHz in dextrose buffer solutions (0.030 S/m and 0.10 S/m). This combined approach included a systematic parametric study to fit a core-shell model to the DEP spectra over the entire tested frequency range, adding robustness to the analysis technique. The membrane capacitance and permittivity were found to be 2.2 pF and 2.0 in 0.030 S/m and 4.5 pF and 4.1 in 0.10 S/m, respectively. Elastin-like polypeptide (ELP-) polyethyleneimine (PEI) copolymer was used to control hMSCs morphology to spheroidal cells and aggregates. Results demonstrated that ELP-PEI treatment controlled hMSCs morphology, increased experiment reproducibility, and concurrently increased hMSCs membrane permittivity to shift the cross-over frequency above 35 MHz. Therefore, ELP-PEI treatment may serve as a tool for the eventual determination of biosurface marker-dependent DEP signatures and hMSCs purification.     

Hydrodynamic mechanisms of cell and particle trapping in microfluidics

Focusing and sorting cells and particles utilizing microfluidic phenomena have been flourishing areas of development in recent years. These processes are largely beneficial in biomedical applications and fundamental studies of cell biology as they provide cost-effective and point-of-care miniaturized diagnostic devices and rare cell enrichment techniques. Due to inherent problems of isolation methods based on the biomarkers and antigens, separation approaches exploiting physical characteristics of cells of interest, such as size, deformability, and electric and magnetic properties, have gained currency in many medical assays. Here, we present an overview of the cell/particle sorting techniques by harnessing intrinsic hydrodynamic effects in microchannels. Our emphasis is on the underlying fluid dynamical mechanisms causing cross stream migration of objects in shear and vortical flows. We also highlight the advantages and drawbacks of each method in terms of throughput, separation efficiency, and cell viability. Finally, we discuss the future research areas for extending the scope of hydrodynamic mechanisms and exploring new physical directions for microfluidic applications.     

Quantitative detection of cells expressing BlaC using droplet-based microfluidics for use in the diagnosis of tuberculosis

This paper describes a method for the quantitative detection of cells expressing BlaC, a β-lactamase naturally expressed by Mycobacterium tuberculosis, intended for the diagnosis of tuberculosis. The method is based on the compartmentalization of bacteria in picoliter droplets at limiting dilutions such that each drop contains one or no cells. The co-encapsulation of a fluorogenic substrate probe for BlaC allows the quantification of bacteria by enumerating the number of fluorescent drops. Quantification of 10 colony forming units per milliliter is demonstrated. Furthermore, the encapsulation of single cell in drops maintains the specificity of the detection scheme even when the concentration of bacteria that do not express BlaC exceeds that expressing BlaC by one million-fold.     

The effect of red blood cell aggregation on velocity and cell-depleted layer characteristics of blood in a bifurcating microchannel

Red blood cell (RBC) aggregation is a multifaceted phenomenon, and whether it is generally beneficial or deleterious remains unclear. In order to better understand its effect on microvascular blood flow, the phenomenon must be studied in complex geometries, as it is strongly dependent on time, flow, and geometry. The cell-depleted layer (CDL) which forms at the walls of microvessels has been observed to be enhanced by aggregation; however, details of the characteristics of the CDL in complex regions, such as bifurcations, require further investigation. In the present study, a microchannel with a T-junction was used to analyze the influence of aggregation on the flow field and the CDL. Micro-PIV using RBCs as tracers provided high resolution cell velocity data. CDL characteristics were measured from the same data using a newly developed technique based on motion detection. Skewed and sharpened velocity profiles in the daughter branches were observed, contrary to the behavior of a continuous Newtonian fluid. RBC aggregation was observed to increase the skewness, but decrease the sharpening, of the velocity profiles in the daughter branches. The CDL width was found to be significantly greater, with a wider distribution, in the presence of aggregation and the mean width increased proportionally with the reciprocal of the fraction of flow entering the daughter branch. Aggregation also significantly increased the roughness of the interface between the CDL and the RBC core. The present results provide further insight into how RBC aggregation may affect the flow in complex geometries, which is of importance in both understanding its functions invivo, and utilizing it as a tool in microfluidic devices.     

Regulation of DNA conformations and dynamics in flows with hybrid field microfluidics

Visualizing single DNA dynamics in flow provides a wealth of physical insights in biophysics and complex flow study. However, large signal fluctuations, generated from diversified conformations, deformation history dependent dynamics and flow induced stochastic tumbling, often frustrate its wide adoption in single molecule and polymer flow study. We use a hybrid field microfluidic (HFM) approach, in which an electric field is imposed at desired locations and appropriate moments to balance the flow stress on charged molecules, to effectively regulate the initial conformations and the deformation dynamics of macromolecules in flow. With λ-DNA and a steady laminar shear flow as the model system, we herein studied the performance of HFM on regulating DNA trapping, relaxation, coil-stretch transition, and accumulation. DNA molecules were found to get captured in the focused planes when motions caused by flow, and the electric field were balanced. The trapped macromolecules relaxed in two different routes while eventually became more uniform in size and globule conformations. When removing the electric field, the sudden stretching dynamics of DNA molecules exhibited a more pronounced extension overshoot in their transient response under a true step function of flow stress while similar behaviors to what other pioneering work in steady shear flow. Such regulation strategies could be useful to control the conformations of other important macromolecules (e.g., proteins) and help better reveal their molecular dynamics.     

Isolation of cells for selective treatment and analysis using a magnetic microfluidic chip

This study describes the development and testing of a magnetic microfluidic chip (MMC) for trapping and isolating cells tagged with superparamagnetic beads (SPBs) in a microfluidic environment for selective treatment and analysis. The trapping and isolation are done in two separate steps; first, the trapping of the tagged cells in a main channel is achieved by soft ferromagnetic disks and second, the transportation of the cells into side chambers for isolation is executed by tapered conductive paths made of Gold (Au). Numerical simulations were performed to analyze the magnetic flux and force distributions of the disks and conducting paths, for trapping and transporting SPBs. The MMC was fabricated using standard microfabrication processes. Experiments were performed with E. coli (K12 strand) tagged with 2.8 μm SPBs. The results showed that E. coli can be separated from a sample solution by trapping them at the disk sites, and then isolated into chambers by transporting them along the tapered conducting paths. Once the E. coli was trapped inside the side chambers, two selective treatments were performed. In one chamber, a solution with minimal nutrition content was added and, in another chamber, a solution with essential nutrition was added. The results showed that the growth of bacteria cultured in the second chamber containing nutrient was significantly higher, demonstrating that the E. coli was not affected by the magnetically driven transportation and the feasibility of performing different treatments on selectively isolated cells on a single microfluidic platform.     

Exploitation of physical and chemical constraints for three-dimensional microtissue construction in microfluidics

There are a plethora of approaches to construct microtissues as building blocks for the repair and regeneration of larger and complex tissues. Here we focus on various physical and chemical trapping methods for engineering three-dimensional microtissue constructs in microfluidic systems that recapitulate the in vivo tissue microstructures and functions. Advances in these in vitro tissue models have enabled various applications, including drug screening, disease or injury models, and cell-based biosensors. The future would see strides toward the mesoscale control of even finer tissue microstructures and the scaling of various designs for high throughput applications. These tools and knowledge will establish the foundation for precision engineering of complex tissues of the internal organs for biomedical applications.     

A study on the glucose uptake,pyruvate and lactate formation in red blood cells of normal,sickle cell trait and sickle cell patients

The rate of glucose consumption and pyruvate and lactate production in red blood cells of normal, sickle cell trait and SS disease subjects were measured. Glucose consumption was found to be increased significantly (p<0.05) in red cells of sickle cell patients than the sickle cell trait and normal persons. There was also an increased rate (p<0.05) of pyruvate formation and a significant (p<0.05) increase in lactate formation in sickle red cells.     

Asymmetry of red blood cell motions in a microchannel with a diverging and converging bifurcation

In microcirculation, red blood cells (RBCs) flowing through bifurcations may deform considerably due to combination of different phenomena that happen at the micro-scale level, such as: attraction effect, high shear, and extensional stress, all of which may influence the rheological properties and flow behavior of blood. Thus, it is important to investigate in detail the behavior of blood flow occurring at both bifurcations and confluences. In the present paper, by using a micro-PTV system, we investigated the variations of velocity profiles of two working fluids flowing through diverging and converging bifurcations, human red blood cells suspended in dextran 40 with about 14% of hematocrit level (14 Hct) and pure water seeded with fluorescent trace particles. All the measurements were performed in the center plane of rectangular microchannels using a constant flow rate of about 3.0 × 10⁻¹² m³/s. Moreover, the experimental data was compared with numerical results obtained for Newtonian incompressible fluid. The behavior of RBCs was asymmetric at the divergent and convergent side of the geometry, whereas the velocities of tracer particles suspended in pure water were symmetric and well described by numerical simulation. The formation of a red cell-depleted zone immediately downstream of the apex of the converging bifurcation was observed and its effect on velocity profiles of RBCs flow has been investigated. Conversely, a cell-depleted region was not formed around the apex of the diverging bifurcation and as a result the adhesion of RBCs to the wall surface was enhanced in this region.     

有机硅油的理化特性及其在医疗保健中的奇特用途

本文浅析了有机硅油具有非凡的抗热、消泡、抗水、抗氧性、化学隋性、生理隋性和柔韧性,展望了有机硅在医疗保健制品中所具有的广泛而奇特的用途。     

Dielectrophoretic discrimination of bovine red blood cell starvation age by buffer selection and membrane cross-linking

We report an interesting buffer electric relaxation time tuning technique, coupled with a glutaraldehyde cross-linking cell fixation reaction, which allows for sensitive dielectrophoretic analysis and discrimination of bovine red blood cell (bRBC) starvation age. The buffer composition is selected such that two easily accessible dielectrophoretic crossover frequencies (cof) exist. Low concentration glutaraldehyde fixation was observed to produce a threefold decrease in the higher cof with a comparable increase in the lower cof also witnessed. More importantly, increased glutaraldehyde fixation concentration significantly increased the higher cof by a factor found to be sensitive to the bRBC starvation age.     

Continuous separation of blood cells in spiral microfluidic devices

Blood cell sorting is critical to sample preparation for both clinical diagnosis and therapeutic research. The spiral inertial microfluidic devices can achieve label-free, continuous separation of cell mixtures with high throughput and efficiency. The devices utilize hydrodynamic forces acting on cells within laminar flow, coupled with rotational Dean drag due to curvilinear microchannel geometry. Here, we report on optimized Archimedean spiral devices to achieve cell separation in less than 8 cm of downstream focusing length. These improved devices are small in size (<1 in.²), exhibit high separation efficiency (∼95%), and high throughput with rates up to 1 × 10⁶ cells per minute. These device concepts offer a path towards possible development of a lab-on-chip for point-of-care blood analysis with high efficiency, low cost, and reduced analysis time.     

Apoptosis in health and disease and modulation of apoptosis for therapy: An overview

Apoptosis a physiological mechanism that eliminates excessive, damaged or unwanted cells, is a highly regulated pathway important for maintaining homeostasis in multicellular organisms. It can be initiated through various signals via the extrinsic pathway which involves death receptors, or via the intrinsic pathway which is initiated by intracellular damage and involves the mitochondria and release of cytochrome c from it to further activate caspases. The Bcl-2 family of proteins is situated upstream to the irreversible damage of cellular constituents and is an important checkpoint in the fate of a cell. The pro-apoptotic members, BH3 only members include BID, BAD and BIM. They directly or indirectly activate multidomain BAX/BAK that constitute the requisite gateway to the intrinsic pathway which operates at the mitochondrial surface and endoplasmic reticulum. In contrast, antiapoptotic members such as Bcl-2, Bcl-XL bind and sequester activation. Downstream of mitochondria, the apoptosome involvement is seen to generate caspase activity. Post mitochondria regulation involves IAPs, and their inhibitors. The pathogenesis of several diseases such as cancer, neurodegenerative disorders, autoimmune disorders, heart disease, infectious diseases including AIDS is closely related to aberrant apoptosis. Consequently interest has emerged in employing various the rapeutic approaches such as gene therapy, antisense therapy, recombinant biologicals, organic and combinatorial chemistry, to specifically target apoptosis signaling pathways such as death receptors FAS/TRAIL, Bcl-2, p53, IAPs, SMAC and caspases, etc. and are now advancing from preclinical to clinical phase.     

A microfluidic chip for direct and rapid trapping of white blood cells from whole blood

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