Debjani Paul

Extracellular vesicles (EVs) are lipid membrane-bound nanoscale (20 nm - 1000 nm) objects shed by all cells. As EVs play an important role in intercellular signaling, these have emerged as promising biomarkers for many diseases including cancer and neurodegenerative disorders. A major bottleneck in research into EVs and their smooth translation to clinic as disease biomarkers is a lack of access to affordable and user-friendly technologies to quickly isolate EVs from complex biological and clinical samples with acceptable purity and yield. Here, we report two different designs of inertial microfluidic devices (e.g. LoAR-Exo and HiAR-Exo) that can isolate EVs from different kinds of biological samples in a single label-free step using a spiral microchannel design with different aspect ratios. LoAR-Exo has a height-to-width aspect ratio ~1, while HiAR-Exo has a height-to-width aspect ratio of 2.5, optimized by COMSOL simulations. We tested both designs by separating polystyrene particles of size < 1 micrometer from a heterogeneous mixture. We then benchmarked the performance of the microfluidic chips against ultracentrifugation and a precipitation kit by isolating EVs from the cell culture-conditioned media (CCM) of MDA-MB-231 cells. We also compared the microchip with SEC by using the CCM from H1975/OR cells. The size distribution of EVs isolated by the microfluidic chip was comparable with ultracentrifugation, precipitation and SEC. In summary, both devices isolated EVs from as little as 1 mL of sample volume using a label-free technique in a continuous manner and without any user intervention. Both microfluidic platforms offer a simple, efficient, and scalable alternative to conventional methods for EV isolation.

Red blood cells (RBCs) become sickle‐shaped and stiff under hypoxia as a consequence of hemoglobin (Hb) polymerization in sickle cell anemia. Distinguishing between sickle cell disease and trait is crucial during the diagnosis of sickle cell disease. While genetic analysis or high‐performance liquid chromatography (HPLC) can accurately differentiate between these two genotypes, these tests are unsuitable for field use. Here, we report a novel microscopy‐based diagnostic test called ShapeDx™ to distinguish between disease and trait blood in less than 1 h. This is achieved by mixing an unknown blood sample with low and high concentrations of a chemical oxygen scavenger and thereby subjecting the blood to slow and fast hypoxia, respectively. The different rates of Hb polymerization resulting from slow and fast hypoxia lead to two distinct RBC shape distributions in the same blood sample, which allows us to identify it as healthy, trait, or disease. The controlled hypoxic environment necessary for differential Hb polymerization is generated using an imaging microchamber, which also reduces the sickling time of trait blood from several hours to just 30 min. In a single‐blinded proof‐of‐concept study conducted on a small cohort of clinical samples, the results of the ShapeDx™ test were 100% concordant with HPLC results. Additionally, our field studies have demonstrated that ShapeDx™ is the first reported microscopy test capable of distinguishing between sickle cell disease and trait samples in resource‐limited settings with the same accuracy as a gold standard test.

We developed a two-layer microfluidic device to study the motility dynamics of E. coli. We show that both confinement and chemoattractant lead to anomalous diffusion, with power-law distributed run lengths and suppression of large-angle tumbles.

SUMMARY: We report a high-throughput microfluidic device to determine the Young’s modulus of single red blood cells (RBCs). Our device consists of a single channel opening into a funnel, with a semi-circular obstacle placed at the mouth of the funnel. As a RBC passes the obstacle, it deflects from its original path. Using populations of artificially-stiffened RBCs, we show that the stiffer RBCs deflect more compared to the healthy RBCs. We then generate a calibration curve that maps each RBC trajectory to its Young’s modulus obtained using an atomic force microscope. Finally, we sort a mixed population of RBCs based on their deformability alone. Our device could potentially be further miniaturized to sort and obtain the elastic constants of nanoscale objects, such exosomes, whose shape change is difficult to monitor by optical microscopy.

Step emulsification (SE), which generates droplets by a sharp change in confinement, has emerged as a potential alternative to flow-focusing technology. Water/dispersed phase is continuously pumped through a shallow inlet channel into a deep chamber pre-filled with the oil/continuous phase. The need for one or more pumps to maintain a continuous flow for droplet generation, and the consequent use of high sample volumes, limit this technique to research labs. Here, we report a pumpfree SE technique for rapid and high-throughput generation of monodisperse hydrogel (agarose) beads using µ l sample volume. Instead of using syringe pumps, we sequentially pipetted oil and liquid agarose into a microfluidic SE device to generate between 20000 and 80000 agarose beads in ∼ 2 min. We also demonstrated the encapsulation of loop-mediated isothermal amplification mixture inside these beads at the time of their formation. Finally, using these beads as reaction chambers, we amplified nucleic acids from P. falciparum and SARS-CoV-2 inside them. The pump-free operation, tiny sample volume, and high-throughput generation of droplets by SE make our technique suitable for point-of-care diagnostics.

Centrifugal microfluidic platforms are becoming increasing popular in many research and diagnostic applications. A major challenge in centrifugal microfluidics is continuous handling of large sample volumes. Keeping the flow rate constant during sample inflow is difficult without a pump. We report an affordable (<USD 40) and portable platform that can handle sample volumes of up to 50 ml without a pump. We use a Mariotte bottle for sample inflow into the disk at a constant flow rate and with a throughput of 1 ml/s. Our pumping mechanism allows basic operations, such as volume metering, flow switching, and mixing. Our platform fulfills the need for portable and affordable instrumentation in developing countries.