Sarkar A, Hou HW, Han J, Alter G.; 2019. System and method for multiplexed affinity purification of proteins and cells. United States of America patent 10228313B2. Publisher's Version
Jennewein MF, Goldfarb I, Dolatshahi S, Cosgrove C, Noelette FJ, Krykbaeva M, Das J, Sarkar A, Gorman MJ, Fischinger S, et al. Fc Glycan-Mediated Regulation of Placental Antibody Transfer. Cell. 2019;178 (1) :202-215.e14.Abstract
Despite the worldwide success of vaccination, newborns remain vulnerable to infections. While neonatal vaccination has been hampered by maternal antibody-mediated dampening of immune responses, enhanced regulatory and tolerogenic mechanisms, and immune system immaturity, maternal pre-natal immunization aims to boost neonatal immunity via antibody transfer to the fetus. However, emerging data suggest that antibodies are not transferred equally across the placenta. To understand this, we used systems serology to define Fc features associated with antibody transfer. The Fc-profile of neonatal and maternal antibodies differed, skewed toward natural killer (NK) cell-activating antibodies. This selective transfer was linked to digalactosylated Fc-glycans that selectively bind FcRn and FCGR3A, resulting in transfer of antibodies able to efficiently leverage innate immune cells present at birth. Given emerging data that vaccination may direct antibody glycosylation, our study provides insights for the development of next-generation maternal vaccines designed to elicit antibodies that will most effectively aid neonates.
Sarkar A, Lu L, Han J, Alter G. Electrical Detection of Glycan Biomarkers for Point-of-Care Diagnosis of Tuberculosis. 2018 Annual Meeting of the Biomedical Engineering Society (BMES2018). 2018.
Sarkar A, Wu L, Han J. Microfluidic Probes for Single Cell Proteomic Analysis. In: Open-Space Microfluidics: Concepts, Implementations, Applications. 1st ed. Wiley-VCH Verlag GmbH & Co. KGaA ; 2018. pp. 221-248. Publisher's Version
Sarkar A, Lu L, Han J, Alter G. An Integrated Electrical Enzyme-linked Immunosorbent Assay (E2LISA) for Point-of-Care Diagnosis of Tuberculosis Using Glycan Biomarkers. 21st International Conference on Miniaturized Systems for Chemistry and Life Sciences (µTAS 2017). 2017.
Sarkar A, Hou HW, Han J, Alter G.; 2016. System and method for multiplexed affinity purification of proteins and cells. WIPO (World Intellectual Prop Org) patent 2016077055 A1. Full Text
Sarkar A, Lofano G, Blackburn S, Han J, Alter G. A Field-Portable, Inertio-Magnetic Microfluidic Cell Sorter for Rapid Discovery of Zika-Specific Antibodies, in 2016 Annual Meeting of the Biomedical Engineering Society (BMES2016). Minneapolis, USA ; 2016.
Sarkar A, Hou HW, Mahan AE, Han J, Alter G. Multiplexed affinity-based separation of proteins and cells using inertial microfluidics. Scientific Reports. 2016;6 :23589. Full TextAbstract

Isolation of low abundance proteins or rare cells from complex mixtures, such as blood, is required for many diagnostic, therapeutic and research applications. Current affinity-based protein or cell separation methods use binary ‘bind-elute’ separations and are inefficient when applied to the isolation of multiple low-abundance proteins or cell types. We present a method for rapid and multiplexed, yet inexpensive, affinity-based isolation of both proteins and cells, using a size-coded mixture of multiple affinity-capture microbeads and an inertial microfluidic particle sorter device. In a single binding step, different targets–cells or proteins–bind to beads of different sizes, which are then sorted by flowing them through a spiral microfluidic channel. This technique performs continuous-flow, high throughput affinity-separation of milligram-scale protein samples or millions of cells in minutes after binding. We demonstrate the simultaneous isolation of multiple antibodies from serum and multiple cell types from peripheral blood mononuclear cells or whole blood. We use the technique to isolate low abundance antibodies specific to different HIV antigens and rare HIV-specific cells from blood obtained from HIV+ patients.

Sarkar A, Hou HW, Han J, Alter G. Integrated Inertial and Magnetic Microfluidic Portable Cell Sorter for Infectious Disease Applications in Resource-Poor Settings, in 2015 Annual Meeting of the Biomedical Engineering Society (BMES2015). Tampa, USA ; 2015.
Wu L, Claas AM, Sarkar A, Lauffenburger DA, Han J. High-throughput protease activity cytometry reveals dose-dependent heterogeneity in PMA-mediated ADAM17. Integrative Biology. 2015;7 :513-524. Full TextAbstract


As key components of autocrine signaling, pericellular proteases, a disintegrin and metalloproteinases (ADAMs) in particular, are known to impact the microenvironment of individual cells and have significant implications in various pathological situations including cancer, inflammatory and vascular diseases. There is great incentive to develop a high-throughput platform for single-cell measurement of pericellular protease activity, as it is essential for studying the heterogeneity of protease response and the corresponding cell behavioral consequences. In this work, we developed a microfluidic platform to simultaneously monitor protease activity of many single cells in a time-dependent manner. This platform isolates individual microwells rapidly on demand and thus allows single-cell activity measurement of both cell-surface and secreted proteases by confining individual cells with diffusive FRET-based substrates. With this platform, we observed dose-dependent heterogeneous protease activation of HepG2 cells treated with phorbol 12-myristate 13-acetate (PMA). To study the temporal behavior of PMA-induced protease response, we monitored the pericellular protease activity of the same single cells during three different time periods and revealed the diversity in the dynamic patterns of single-cell protease activity profile upon PMA stimulation. The unique temporal information of single-cell protease response can help unveil the complicated functional role of pericellular proteases.

Sarkar A, Hou HW, Mahan AE, Han J, Alter G. Inertial Microfluidics for Multiplexed Affinity Separation of Proteins and Cells, in 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences (μTAS 2014). San Antonio, USA ; 2014. Full Text
Cheow LF, Sarkar A, Kolitz S, Lauffenburger DA, Han J. Detecting Kinase Activities from Single Cell Lysate Using Conentration-Enhanced Mobility Shift Assay. Analytical Chemistry. 2014;86 (15) :7455-7462. Full TextAbstract

Electrokinetic preconcentration coupled with mobility shift assays can give rise to very high detection sensitivities. We describe a microfluidic device that utilizes this principle to detect cellular kinase activities by simultaneously concentrating and separating substrate peptides with different phosphorylation states. This platform is capable of reliably measuring kinase activities of single adherent cells cultured in nanoliter volume microwells. We also describe a novel method utilizing spacer peptides that significantly increase separation resolution while maintaining high concentration factors in this device. Thus, multiplexed kinase measurements can be implemented with single cell sensitivity. Multiple kinase activity profiling from single cell lysate could potentially allow us to study heterogeneous activation of signaling pathways that can lead to multiple cell fates.

Microfluidic Probe for Single Cell Analysis in Adherent Tissue Culture
Sarkar A*, Kolitz S*, Lauffenburger DA, Han J. Microfluidic Probe for Single Cell Analysis in Adherent Tissue Culture. Nature Communications. 2014;5 :3421. Full TextAbstract
Single-cell analysis provides information critical to understanding key disease processes that are characterized by significant cellular heterogeneity. Few current methods allow single-cell measurement without removing cells from the context of interest, which not only destroys contextual information but also may perturb the process under study. Here we present a microfluidic probe that lyses single adherent cells from standard tissue culture and captures the contents to perform single-cell biochemical assays. We use this probe to measure kinase and housekeeping protein activities, separately or simultaneously, from single human hepatocellular carcinoma cells in adherent culture. This tool has the valuable ability to perform measurements that clarify connections between extracellular context, signals and responses, especially in cases where only a few cells exhibit a characteristic of interest.
Chen C-H, Miller MA, Sarkar A, Beste MT, Isaacson KB, Lauffenburger DA, Griffith LG, Han J. Multiplexed Protease Activity Assay for Low Volume Clinical Samples Using Droplet Based Microfluidics and Its Application to Endometriosis. J Am Chem Soc. 2013;135 (5) :1645-48. Full TextAbstract
As principal degrading enzymes of the extracellular matrix, metalloproteinases (MPs) contribute to various pathologies and represent a family of promising drug targets and biomarker candidates. However, multiple proteases and endogenous inhibitors interact to govern MP activity, often leading to highly context-dependent protease function that unfortunately has impeded associated clinical utility. We present a method for rapidly assessing the activity of multiple specific proteases in small volumes (<20 μL) of complex biological fluids such as clinical samples that are available only in very limited amounts. It uses a droplet-based microfluidic platform that injects the sample into thousands of picoliter-scale droplets from a barcoded droplet library (DL) containing mixtures of unique, moderately selective FRET-based protease substrates and specific inhibitors and monitors hundreds of the reactions thus initiated simultaneously by tracking these droplets. Specific protease activities in the sample are then inferred from the reaction rates using a deconvolution technique, proteolytic activity matrix analysis (PrAMA). Using a nine-member DL with three inhibitors and four FRET substrates, we applied the method to the peritoneal fluid of subjects with and without the invasive disease endometriosis. The results showed clear and physiologically relevant differences with disease, in particular, decreased MMP-2 and ADAM-9 activities.
Li LD, Crouzier T, Sarkar A, Dunphy L, Han J, Ribbeck K. Spatial Configuration and Composition of Charge Modulates Transport into a Mucin Hydrogel Barrier. Biophys J. 2013;105 (6) :1357-65. Full TextAbstract
The mucus barrier is selectively permeable to a wide variety of molecules, proteins, and cells, and establishes gradients of these particulates to influence the uptake of nutrients, the defense against pathogens, and the delivery of drugs. Despite its importance for health and disease, the criteria that govern transport through the mucus barrier are largely unknown. Studies with uniformly functionalized nanoparticles have provided critical information about the relevance of particle size and net charge for mucus transport. However, these particles lack the detailed spatial arrangements of charge found in natural mucus-interacting substrates, such as certain viruses, which may have important consequences for transport through the mucus barrier. Using a novel, to our knowledge, microfluidic design that enables us to measure real-time transport gradients inside a hydrogel of mucins, the gel-forming glycoprotein component of mucus, we show that two peptides with the same net charge, but different charge arrangements, exhibit fundamentally different transport behaviors. Specifically, we show that certain configurations of positive and negative charges result in enhanced uptake into a mucin barrier, a remarkable effect that is not observed with either charge alone. Moreover, we show that the ionic strength within the mucin barrier strongly influences transport specificity, and that this effect depends on the detailed spatial arrangement of charge. These findings suggest that spatial charge distribution is a critical parameter to modulate transport through mucin-based barriers, and have concrete implications for the prediction of mucosal passage, and the design of drug delivery vehicles with tunable transport properties.
Sarkar A, Kolitz S, Lauffenburger DA, Han J. An Integrated Microfluidic Probe for Multiplexed Single Cell Kinase Activity Measurement, in International Conference on the Systems Biology of Human Disease (SBHD2012). Heidelberg, Germany ; 2012.
Chen C-H, Miller MA, Sarkar A, Beste MT, Lauffenburger DA, Griffith LG, Han J. High-Throughput Multiplexed Protease Activity Measurement using a Droplet Based Microfluidic Platform with Picoinjector. 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences (µTAS 2012). 2012. Full Text
Sarkar A, Kolitz S, Lauffenburger, Douglas A., Han J. An Integrated Microfluidic Probe for Multiplexed Single Cell Kinase Activity Measurement. 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences (µTAS 2012). 2012 :416-418. Full TextAbstract

We present an integrated microfluidic probe that enables the measurement of multiple kinase activities in selected phenotypically distinct single cells from large cell populations on standard tissue culture platforms. The contents of a cell are captured without disturbing its extracellular context by creating a small lysis zone at the probe tip by hydrodynamic confinement. Pneumatic micro-valves are then used to separate and mix the captured lysate into different assay mixtures in separate small volume chambers for a fluorimetric assay. We demonstrate here the ability to simultaneously measure the activity of three kinases: Akt, MAPKAPK2, PKA and a loading control enzyme, GAPDH, from a single cell. This single cell assay platform enables the correlation of cellular phenotype to intracellular biochemical state at the single cell level and hence can provide a clearer understanding of cell behavior in heterogeneous cell populations.

Chen C, Sarkar A, Song Y, Miller MA, Kim SJ, Griffith LG, Lauffenburger DA, Han J. Enhancing Protease Activity Assay in Droplet-Based Microfluidics Using a Biomolecule Concentrator, in 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences (μTAS 2011). Seattle, USA ; 2011. Full Text
Sarkar A, Kolitz S, Cheow LF, Lauffenburger DA, Han J. An Integrated Microfluidic Probe for Concentration-Enhanced Selective Single Cell Kinase Activity Measurement. 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences (µTAS 2011). 2011 :1394-1396. Full TextAbstract
We present an integrated microfluidic probe that captures the contents of selected single cells from a cell population adhe-rent on a standard tissue culture platform and directly measures specific protein kinase activities in the captured single cell ly-sate. A kinetic fluorimetric kinase assay in a small integrated chamber isolated by micro-valves and an end-point concentra-tion-enhanced mobility-shift kinase assay in an integrated nanofluidic concentrator are demonstrated. We demonstrate the use of the probe by measuring MAPKAPK2 and AKT kinase activity in single human hepatocellular carcinoma (HepG2) cells.