Research
Hyper-throughput Microfluidics
Ultrahigh-throughput sorting of rare circulating tumor cells (CTCs) from leukapheresis products
CTC-based liquid biopsies have emerged as a promising tool for cancer diagnostics, treatment selection, and response monitoring. However, despite impressive technological gains, the presence of only a few CTCs in the standard 10 mL blood samples has severely limited their clinical utility. Leukapheresis products (leukopaks) obtained by screening whole human blood (~5 liters) can enhance the number of isolated CTCs to thousands of cells (100-fold). Even though leukopaks provide a path to isolating a large number of CTCs, owing to their large volume (~120 mL) and the presence of 100-fold more nucleated cells than in a 10 mL blood sample, current state-of-the-art cell sorting techniques can only process 3-5% of the sample. We developed an ultrahigh-throughput LPCTC-iChip cell sorting platform to process the full leukopak volume and recover thousands of viable CTCs.
Ultrahigh-throughput magnetic sorting of large blood volumes for epitope-agnostic isolation of circulating tumor cells, A. Mishra, T. Dubash et al., PNAS, 2020. https://doi.org/10.1073/pnas.2006388117.
Microfluidic concentration and separation of circulating tumor cell clusters from large blood volumes, J. F. Edd, A. Mishra et al., Lab on a Chip, 2020. https://doi.org/10.1039/C9LC01122F
High-throughput Microfluidic Devices for Cell and Gene Therapy Manufacturing
Boosted by a 5-year K-25 career award from NIH (2023-2028), we will develop an efficient microfluidic technology for stem cell isolation from blood products of patients with Sickle Cell Disease (SCD). SCD is a genetic disorder that affects ~100,000 Americans in the United States alone. Obtaining a sufficient number of blood stem cells is paramount to the success of stem cell gene therapy. However, the higher numbers of sickled RBCs adversely hamper the yield of CD34+ stem cells during purification, leading to the loss of ~50% of highly valuable stem cell dose. There is, therefore, an immediate need to develop an isolation technology that can recover stem cells from full leukopaks with high yield. We will develop a microfluidic stem cell isolation technology (HSPC-iChip) that can recover CD34+ cells from full apheresis products while depleting contaminating cells.
Optoelectrical Tweezers: Rapid Electrokinetic Patterning (REP)
REP research highlighted on the cover of the Trends in Biotechnology Journal (Cell Press)
Rapid Electrokinetic Patterning (REP), developed in Wereley Group at Purdue University, is one of the emerging particle manipulation technique. Using a laser-induced AC electrothermal flow and short range particle-electrode interactions, REP can capture and sort nano- to microparticles at the focal point of the laser on an electrode surface. The trapped entities can be manipulated precisely and accurately on the electrode surface by simply moving the laser spot.
For more information on REP, please read "Optoelectrical microfluidics as a promising tool in biology," A. Mishra, J.-S. Kwon, R. Thakur and S. Wereley, Trends in Biotechnology, Cell Press, 2014 (*Cover Article). http://dx.doi.org/10.1016/j.tibtech.2014.06.002
Trapping swimming bacteria using optoelectrical tweezers
Enterobacter aerogenes bacteria in a REP trap
In previous studies, REP has been used to capture non-swimming bacteria or inert polystyrene particles. In this project, I demonstrated trapping of highly motile swimming bacteria, relevant to chemotaxis studies and probed effect of REP trapping environment on the viability of cells by using SYTO9/PI staining. I studied the individual and combined effect of the optical radiation, laser-induced heating, electric field and shear forces on membrane integrity of the trapped cells. The experimental results provided invaluable insights into the safe parameters for the REP trapping of swimming microorganisms.
"Trapping and viability of swimming bacteria in an optoelectric trap," A. Mishra, T. Maltais, T. Walter, A. Wei, S. Williams and S. Wereley, Lab on a Chip, 2016. http://dx.doi.org/10.1039/C5LC01559F
Optoelectrical Tweezers Physics
An illustration of the side-view microscopy experimental setup
By analyzing the trajectory of trapped particles with subpixel resolution, we show that the transverse trapping force in REP originates due to axisymmetric Stokes drag experienced by the particles in toroidal electrothermal vortices. The trapping force scales linearly with radial distance from the trap center and trap stiffness is on the order of femtonewtons/μm in the transverse plane. This low trap stiffness would be useful for measuring femtonewton-scale forces. Additionally, in previous REP applications, 350 to 700 nm indium tin oxide (ITO) layers have been used as electrodes. Using theoretical, computational and side-view micro-particle Image velocimetry methods, I showed that Ti is a better electrode material as it can creates an AC electrothermal flow of the same speed while requiring only a fraction of the optical radiation used by ITO.
"Nature of trapping forces in optically induced electrothermal vortex-based tweezers," A. Mishra, K. Gupta, and S. Wereley, Physical Review Fluids, 2021. https://doi.org/10.1103/PhysRevFluids.6.023701
"Optoelectric patterning: Effect of electrode material and thickness on laser-induced AC electrothermal flow," A. Mishra, J.-W. Khor, K. Clayton, S. Williams, X. Pan, T. Kinzer-Ursem and S. Wereley, Electrophoresis, 2015. http://dx.doi.org/10.1002/elps.201500473
Nano Tweezers
Patterning CNTs using REP
On-demand trapping, manipulation and deposition of vertically oriented carbon nanotubes and plasmonic nanostructures for REP action
Ability to position a single or multiple CNTs in a vertical orientation on an electrode surface at room temperature can allow us to fabricate highly complex bottom-up CNT-based structures. In this work, we showed that REP can be used to trap a single or multiple CNTs which can be manipulated to any desired spot on the electrode surface where the trapped nanotubes can be electrophoretically deposited by spiking the AC electric field with a DC offset. We also demonstrate that the number of nanotubes in a REP trap can be dynamically tuned by changing AC frequency or the concentration of nanotubes, thus allowing us to control not only the place of deposition but also the number of nanotubes. Additionally, In a close collaboration with a leading Nanophotonics Lab at Purdue, we explored the use of resonant gold nanodiscs on a silicon substrate for subwavelength confinement of nanoparticles and viruses.
"Photothermal heating enabled by plasmonic nanostructures for electrokinetic manipulation and sorting of particles," J. C. Ndukaife, A. Mishra, U. Guler, A. A. Nnanna, S. Wereley and A. Boltasseva, ACS Nano, 2014. http://dx.doi.org/10.1021/nn502294w
"Dynamic optoelectric trapping and deposition of multiwalled carbon nanotubes," A. Mishra, K. Clayton, V. Velasco, S. Williams and S. Wereley, Nature Microsystems & Nanoengineering, Nature Publishing Group, 2016. http://dx.doi.org/10.1038/micronano.2016.5
Confined Plunging Liquid Jet experimental setup
Confined Plunging Liquid Jet
Investigation of bubble plume dispersion in confined plunging liquid jets (CPLJ)
MSc Thesis Project, Department of Engineering and Applied Science, Cranfield University, UK and Fachpraktikum at HZDR, Germany
Air entrainment by an impinging liquid jet is a phenomenon of high industrial importance. One prominent application is found in the Nuclear Safety Research. During a loss of coolant accident (LOCA) in a power plant, the emergency core cooling (ECC) is activated. In some accident scenarios, the cold water is injected into the cold leg which is partially filled with hot water and steam. For the integrity of the reactor pressure vessel (RPV), sufficient mixing of cold and hot water needs to be assured in order to avoid thermal shock. The CPLJ system can also be used as an oxygen transfer device in the wastewater treatment. In this work, a confined plunging liquid jet system was experimentally and numerically analyzed. On experimental front, a laboratory scale CPLJ setup was developed. The impact of the change in jet height, water flow rate, and nozzle diameter on bubble plume size and surrounding flow field was investigated through high-speed camera photography and Particle Image Velocimetry. The objective of the work was the simultaneous measurement of the liquid and bubble velocities using Particle Image Velocimetry and Particle Tracking Velocimetry. On the numerical front, a 3D Euler-Euler two-fluid CFD simulation of bubble plume dispersion was performed with the CFD software ANSYS CFX 12.1.
This work was also presented at the International Conference on Multiphase Flow (ICMF 2010), Florida.