Ali Asgar S. Bhagat

Isolation and retrieval of circulating tumor cells using centrifugal forces

Presence and frequency of rare circulating tumor cells (CTCs) in bloodstreams of cancer patients are pivotal to early cancer detection and treatment monitoring. Here, we use a spiral microchannel with inherent centrifugal forces for continuous, size-based separation of CTCs from blood (Dean Flow Fractionation (DFF)) which facilitates easy coupling with conventional downstream biological assays. Device performance was optimized using cancer cell lines (> 85% recovery), followed by clinical validation with positive CTCs enumeration in all samples from patients with metastatic lung cancer (n = 20; 5–88u2005CTCs per mL). The presence of CD133+ cells, a phenotypic marker characteristic of stem-like behavior in lung cancer cells was also identified in the isolated subpopulation of CTCs. The spiral biochip identifies and addresses key challenges of the next generation CTCs isolation assay including antibody independent isolation, high sensitivity and throughput (3u2005mL/hr); and single-step retrieval of viable CTCs.

Microfluidics for cell separation.

The need for efficient cell separation, an essential preparatory step in many biological and medical assays, has led to the recent development of numerous microscale separation techniques. This review describes the current state-of-the-art in microfluidics-based cell separation techniques. Microfluidics-based sorting offers numerous advantages, including reducing sample volumes, faster sample processing, high sensitivity and spatial resolution, low device cost, and increased portability. The techniques presented are broadly classified as being active or passive depending on the operating principles. The various separation principles are explained in detail along with popular examples demonstrating their application toward cell separation. Common separation metrics, including separation markers, resolution, efficiency, and throughput, of these techniques are discussed. Developing efficient microscale separation methods that offering greater control over cell population distribution will be important in realizing true point-of-care (POC) lab-on-a-chip (LOC) systems.

Spiral microchannel with rectangular and trapezoidal cross-sections for size based particle separation

The paper reports a new method for three-dimensional observation of the location of focused particle streams along both the depth and width of the channel cross-section in spiral inertial microfluidic systems. The results confirm that particles are focused near the top and bottom walls of the microchannel cross-section, revealing clear insights on the focusing and separation mechanism. Based on this detailed understanding of the force balance, we introduce a novel spiral microchannel with a trapezoidal cross-section that generates stronger Dean vortices at the outer half of the channel. Experiments show that particles focusing in such device are sensitive to particle size and flow rate, and exhibits a sharp transition from the inner half to the outer half equilibrium positions at a size-dependent critical flow rate. As particle equilibration positions are well segregated based on different focusing mechanisms, a higher separation resolution is achieved over conventional spiral microchannels with rectangular cross-section.

Microfluidic Devices for Blood Fractionation

Abstract: Blood, a complex biological fluid, comprises 45% cellular components suspended in protein rich plasma. These different hematologic components perform distinct functions in vivo and thus the ability to efficiently fractionate blood into its individual components has innumerable applications in both clinical diagnosis and biological research. Yet, processing blood is not trivial. In the past decade, a flurry of new microfluidic based technologies has emerged to address this compelling problem. Microfluidics is an attractive solution for this application leveraging its numerous advantages to process clinical blood samples. This paper reviews the various microfluidic approaches realized to successfully fractionate one or more blood components. Techniques to separate plasma from hematologic cellular components as well as isolating blood cells of interest including certain rare cells are discussed. Comparisons based on common separation metrics including efficiency (sensitivity), purity (selectivity), and

Isoporous Micro/Nanoengineered Membranes

Isoporous membranes are versatile structures with numerous potential and realized applications in various fields of science such as micro/nanofiltration, cell separation and harvesting, controlled drug delivery, optics, gas separation, and chromatography. Recent advances in micro/nanofabrication techniques and material synthesis provide novel methods toward controlling the detailed microstructure of membrane materials, allowing fabrication of membranes with well-defined pore size and shape. This review summarizes the current state-of-the-art for isoporous membrane fabrication using different techniques, including microfabrication, anodization, and advanced material synthesis. Various applications of isoporous membranes, such as protein filtration, pathogen isolation, cell harvesting, biosensing, and drug delivery, are also presented.

Separation of Leukocytes from Blood Using Spiral Channel with Trapezoid Cross-Section

Inertial microfluidics has recently drawn wide attention as an efficient, high-throughput microfluidic cell separation method. However, the achieved separation resolution and throughput, as well as the issues with cell dispersion due to cell-cell interaction, have appeared to be limiting factors in the application of the technique to real-world samples such as blood and other biological fluids. In this paper, we present a novel design of a spiral inertial microfluidic (trapezoidal cross-section) sorter with enhanced separation resolution and demonstrate its ability in separating/recovering polymorphonuclear leukocytes (PMNs) and mononuclear leukocytes (MNLs) from diluted human blood (1-2% hematocrit) with high efficiency (>80%). PMNs enriched by our method also showed negligible activation as compared to original input sample, while the conventional red blood cell (RBC) lysis method clearly induced artificial activation of the sensitive PMNs. Therefore, our proposed technique would be a promising alternative to enrich/separate sensitive blood cells for therapeutic or diagnostic applications.

A microfluidics approach towards high-throughput pathogen removal from blood using margination

Sepsis is an adverse systemic inflammatory response caused by microbial infection in blood. This paper reports a simple microfluidic approach for intrinsic, non-specific removal of both microbes and inflammatory cellular components (platelets and leukocytes) from whole blood, inspired by the invivo phenomenon of leukocyte margination. As blood flows through a narrow microchannel (20u2009×u200920u2009µm), deformable red blood cells (RBCs) migrate axially to the channel centre, resulting in margination of other cell types (bacteria, platelets, and leukocytes) towards the channel sides. By using a simple cascaded channel design, the blood samples undergo a 2-stage bacteria removal in a single pass through the device, thereby allowing higher bacterial removal efficiency. As an application for sepsis treatment, we demonstrated separation of Escherichia coli and Saccharomyces cerevisiae spiked into whole blood, achieving high removal efficiencies of ∼80% and ∼90%, respectively. Inflammatory cellular components were also depleted by >80% in the filtered blood samples which could help to modulate the host inflammatory response and potentially serve as a blood cleansing method for sepsis treatment. The developed technique offers significant advantages including high throughput (∼1u2009ml/h per channel) and label-free separation which allows non-specific removal of any blood-borne pathogens (bacteria and fungi). The continuous processing and collection mode could potentially enable the return of filtered blood back to the patient directly, similar to a simple and complete dialysis circuit setup. Lastly, we designed and tested a larger filtration device consisting of 6 channels in parallel (∼6u2009ml/h) and obtained similar filtration performances. Further multiplexing is possible by increasing channel parallelization or device stacking to achieve higher throughput comparable to convectional blood dialysis systems used in clinical settings.

Rapid mixing of sub-microlitre drops by magnetic micro-stirring

We demonstrate rapid mixing of sub-microlitre droplets (250 nl) using miniaturized magnetic stir bars (400 μm × 200 μm × 15 μm). The stir bars are fabricated using laser micromachining and placed on the substrate on which the drops are manipulated. They are activated by an externally applied magnetic field and used in combination with on-demand drop merging in enthalpy arrays. This technique results in a 10-fold increase in mixing rate, and a mixing time constant of about 2 s. Drop mixing times are measured by Förster resonance energy transfer (FRET) and verified by thermodynamic measurements of binding and enzymatic reactions.

Real-time control of a microfluidic channel for size-independent deformability cytometry

Mechanical properties of cells can be correlated with various cell states and are now considered as an important class of biophysical markers. Effectiveness of existing high-throughput microfluidic techniques for investigating cell mechanical properties is adversely affected by cell-size variation in a given cell population. In this work, we introduce a new microfluidic system with real-time feedback control to evaluate single-cell deformability while minimizing cell-size dependence of the measurement. Using breast cancer cells (MCF-7), we demonstrate the potential of this system for stiffness profiling of cells in complex, diverse cell populations.

Deformability Based Cell Margination – A Simple Microfluidic Design for Malarial Infected Red Blood Cell Filtration

In blood vessels with luminal diameter less than 300μm, red blood cells (RBCs) which are smaller in size and more deformable than leukocytes, tend to migrate to the axial centre of the vessel due to the Poiseuille nature of flow within these small capillaries, thus displacing the larger (and less deformable) leukocytes to the vessel wall; a phenomenon aptly termed as margination. In this work, this physiological event is mimicked in microfluidic systems for the biological separation of malarial infected RBCs (iRBCs) from whole blood. Change in cell stiffness is a characteristic of iRBCs which can act as an intrinsic biomarker for separation. Tests were conducted using early ring stage and late trophozoite/schizont stage iRBCs which vary significantly in their deformability. Filtration efficiency was quantified by analyzing the dispersion of these fluorescently labeled microbeads and iRBCs across the microchannel width at the outlet. Flow cytometry analysis was also conducted on the outlet samples to confirm filtration results. Our results indicate filtration efficiency of 75% for early ring stage iRBCs and >90% for late stage iRBCs. This is the first demonstration applying this unique biomimetic separation technique to iRBCs filtration for disease diagnostic application. The simple and passive operation of the system makes it ideal for on-site testing in resource poor settings and can be readily applied to other blood cell diseases such as sickle cell anemia and leukemia which are also characterized by change in cell stiffness.