Microfluidic concentration-enhanced single cell enzyme activity assay

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2013.Cataloged from PDF version of thesis.Includes bibliographical references.Cells sense stimuli, process information and respond using signaling networks regulated by enzymatic activity of various proteins. Aberrations in signaling are associated with diseases such as cancer. Most current methods lack the sensitivity to measure enzymatic activity in single cells and instead measure the average of large cell populations. Cellular heterogeneity, overlooked in these methods, is widespread and relevant. Microfabricated tools are uniquely suited to single cell analysis due to the match in size scale which enables high sensitivity, high throughput measurements. In this thesis we develop a microfluidic platform for the direct measurement of enzyme activities from selected single cells without disrupting their extracellular context. We develop modules to: enhance enzyme assay sensitivity by microfluidic confinement, interface microfluidic devices with selected single cells, enable multiplexing and then integrate these modules to perform single cell assays. We first investigate electrokinetic trapping of charged biomolecules in a nanofluidic concentrator for enhancing enzyme assay sensitivity by simultaneously accumulating enzyme and substrate into a reaction plug. Non-linear enhancement of reaction kinetics in this device is predicted by a mathematical model and experimentally verified. A linear enhancement mode is developed where only the enzyme is accumulated and is reacted with substrate later in an enclosed volume defined by integrated pneumatic valves or by micro-droplets formed using an integrated droplet generator. This device is then used to perform high-throughput measurement of secreted cellular proteases. We then develop a nicrofluidic probe for lysis and capture of the contents of selected single adherent cells from standard tissue culture platforms by creating a small lysis zone at its tip using hydrodynamic confinement. The single cell lysate is then divided and mixed with different substrates and confined in small chambers for fluorimetric assays. An integrated nanofluidic concentrator enables further concentration-enhancement. We demonstrate the ability to measure, from selected single cells, the activity of kinases: Akt, MAPKAPK2, PKA and a metabolic enzyme, GAPDH - separately or simultaneously. This assay platform can correlate single cell phenotype or extracellular context to intracellular biochemical state. We present preliminary explorations of the correlation of cell morphology or local cell population density to kinase activity.by Aniruddh Sarkar.Ph.D

Enhancing Protease Activity Assay in Droplet-Based Microfluidics Using a Biomolecule Concentrator

We introduce an integrated microfluidic device consisting of a biomolecule concentrator and a microdroplet generator, which enhances the limited sensitivity of low-abundance enzyme assays by concentrating biomolecules before encapsulating them into droplet microreactors. We used this platform to detect ultralow levels of matrix metalloproteinases (MMPs) from diluted cellular supernatant and showed that it significantly (~10-fold) reduced the time required to complete the assay and the sample volume used.National Institutes of Health (U.S.) (Grant GM68762)National Institutes of Health (U.S.) (Grant U54-CA112967)National Institutes of Health (U.S.) (Grant R01-EB010246)National Institutes of Health (U.S.) (Grant R01-GM081336)National Science Foundation (U.S.) (Graduate Fellowship)United States. Defense Advanced Research Projects Agency (Cipher Program

Microscale Tools for Biomarker Discovery and Electronic Point-of-Care Diagnostics for Infectious Diseases

Presented on May 1, 2020 from 11:00 a.m.-12:00 p.m. online. Spring 2020 NANOFANS Webinar Series: Session 1.2020 Spring NanoFANS (Focusing on Advanced Nanobio Systems) program will be offered in a weekly webinar format during the month of May 2020. The focus of this event will be “Nanotechnology in Infectious Diseases (Diagnostics/Therapeutics)."Spring 2020 NANOFANS Webinar Series: Session 1. In the current global pandemic situation, infectious diseases are the leading cause of mortality worldwide, with viruses such as, ebola, SARS-Cov, SARS-Cov-2 in particular, making global impact on healthcare and socio-economic development. The rapid development of drug resistance to currently available therapies and associated side effects leads to serious public health concern; hence, devising novel treatment strategies is of paramount importance. The application of nanotechnology in infectious diseases is fast-revolutionizing the biomedical field and the healthcare sector and has a potential to diagnose, treat and prevent diseases." Aniruddh Sarkar is an Assistant Professor in the Wallace H. Coulter Department of Biomedical Engineering at the Georgia Institute of Technology and Emory University where he leads the Micro/Nano Bioelectronics Lab. He was earlier a Research Fellow at the Ragon Institute of MGH, MIT and Harvard with research affiliations at Harvard Medical School and at MIT. His research has evolved around the theme of exploiting unique physical phenomena that occur at the micrometer to nanometer length scales to develop devices and systems for solving various technological problems with a special focus on applications in biology and medicine. His earlier work, with Prof. Galit Alter (MGH/HMS) and Prof. Jongyoon Han (MIT), involved the development and application of microfabricated and nanofabricated devices to further the prevention, diagnosis and therapy of infectious diseases such as Tuberculosis and HIV/AIDS. He received his Ph.D. in Electrical Engineering and Computer Science with a minor in Biology at MIT, developing microfluidic tools for single-cell analysis. He received his bachelors and master’s degrees, both in Electrical Engineering at IIT Bombay.Aniruddh Sarkar is an Assistant Professor in the Wallace H. Coulter Department of Biomedical Engineering at the Georgia Institute of Technology and Emory University where he leads the Micro/Nano Bioelectronics Lab. He was earlier a Research Fellow at the Ragon Institute of MGH, MIT and Harvard with research affiliations at Harvard Medical School and at MIT. His research has evolved around the theme of exploiting unique physical phenomena that occur at the micrometer to nanometer length scales to develop devices and systems for solving various technological problems with a special focus on applications in biology and medicine. His earlier work, with Prof. Galit Alter (MGH/HMS) and Prof. Jongyoon Han (MIT), involved the development and application of microfabricated and nanofabricated devices to further the prevention, diagnosis and therapy of infectious diseases such as Tuberculosis and HIV/AIDS. He received his Ph.D. in Electrical Engineering and Computer Science with a minor in Biology at MIT, developing microfluidic tools for single-cell analysis. He received his bachelors and master’s degrees, both in Electrical Engineering at IIT Bombay.Runtime: 52:15 minutesCurrent worldwide challenges in scaling COVID19 diagnosis underscore the need for developing inexpensive point-of-care diagnostics for infectious diseases. The heterogeneity of the disease – a large number of mild or asymptomatic cases coupled with the rapid degradation in symptoms in some patients – pose a challenge for the healthcare system and emphasize the need for developing predictive biomarkers of disease severity. We are harnessing microscale technology to solve these challenges by developing devices for high-throughput discovery and inexpensive electronic detection of biomarkers. Here, I will present our progress with these approaches – in the context of Tuberculosis and other infectious diseases – and end by outlining our current work in applying them to COVID19 diagnosis and prognostic monitoring

Micro-/Nano-scale Tools for Biomarker Discovery and Electronic Point-of-Care Diagnostics for Infectious Diseases

Presented on April 26, 2022 from 12:00 p.m.-1:00 p.m. in the Marcus Nanotechnology Building, Rooms 1116-1118, Georgia Tech, Atlanta, GA.Aniruddh Sarkar is an Assistant Professor in the Wallace H. Coulter Department of Biomedical Engineering at the Georgia Institute of Technology and Emory University where he leads the Micro/Nano Bioelectronics Lab. His research has evolved around the theme of exploiting unique physical phenomena that occur at the micrometer to nanometer length scales to develop devices and systems for solving various technological problems with a special focus on applications in biology and medicine. This includes the development and application of microfabricated and nanofabricated devices for the prevention, diagnosis and therapy of infectious diseases such as COVID-19 and Tuberculosis. He was earlier a Research Fellow at the Ragon Institute of MGH, MIT, and Harvard with research affiliations at Harvard Medical School and at MIT. He received his Ph.D. in electrical engineering and computer science with a minor in biology at MIT, developing microfluidic tools for single-cell analysis. He received his bachelors and master’s degrees, both in electrical engineering at IIT Bombay.Runtime: 57:42 minutesThe current COVID-19 pandemic and other recent outbreaks such as Ebola, MERS, SARS, and H1N1 have underscored the need for early detection and continued surveillance of emerging and re-emerging infectious diseases. The heterogeneity of disease in COVID-19 – a large number of mild or asymptomatic cases coupled with the relatively rapid degradation in symptoms in some patients – poses a unique challenge for the healthcare system and emphasizes the need for developing predictive biomarkers of disease severity. We are harnessing microscale and nanoscale technology to solve these challenges by developing devices for high-throughput discovery and inexpensive electronic detection of diagnostic & prognostic biomarkers. Here, I will present our progress with these approaches in the context of COVID-19 and beyond

Microfluidic probe for single-cell analysis in adherent tissue culture

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.National Institutes of Health (U.S.) (Grant P50-GM068762)United States. Army Research Office (Institute for Collaborative Biotechnologies Grant W911NF-09-0001

Image analysis aided freshness classification of pool barb fish (Puntius sophore)

Fish is a very nutritious dish that is consumed worldwide as a complete meal. This causes a rise in fish production and storage. Freshness of fish that is stored in ice boxes and deep freezers can degrade very quickly. A stale fish can cause great harm to a human ingesting it as it may carry many diseases. This paper aims to present a hybrid model that can recognise Puntius (commonly Puti Fish) as fresh or stale by using an image and certain values graded on scale of 10 like color, texture, etc. This mainly aims to a non-destructive approach to classify fishes as fresh or stale. The model comprises of a CNN for processing images and a Dense network that extracts information from the numerical data. These features are combined and are then further passed onto another dense neural network that performs the final classification task. We were able to achieve 96–98% accuracy with this model

Multiplexed Affinity-Based Separation of Proteins and Cells Using Inertial Microfluidics

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.United States. Defense Advanced Research Projects Agency (DARPA Dialysis-like Therapy (DLT) program under SSC Pacific N66001-11-1-4182)Bill & Melinda Gates Foundatio

Detecting Kinase Activities from Single Cell Lysate Using Concentration-Enhanced Mobility Shift Assay

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.National Institutes of Health (U.S.) (Grant P50-GM068762)Singapore. Agency for Science, Technology and Research (National Science Scholarship

High-throughput protease activity cytometry reveals dose-dependent heterogeneity in PMA-mediated ADAM17 activation

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.National Institutes of Health (U.S.) (Grant R01-CA096504)Singapore-MIT Alliance for Research and Technology (SMART

High-Throughput Mutiplexed Protease Activity Measurement Using a Droplet Based Microfluidic Platform with Picoinjector

In this study, we integrated several components, including a droplet generator, a pico-injector, and an analytical inference technique, Proteolytic Activity Matrix Analysis (PrAMA), to create a platform for assessing multiple specific protease activity assays with minimal liquid handling and sample-requirement for personal medicine analysis. The microfluidic platform enables the direct measurements of protease enzyme activity, which is more physiologically informative than the standard measurements to determine the enzyme concentration alone. By tracking hundreds of picoliter droplets containing biological samples mixed with unique FRET-based protease substrates and inhibitors, the assay simultaneously infers multiple specific protease activities with minimal (<20µl) physiological sample requirement. We applied this method to in vitro study of an immortalized cell line established from a peritoneal endometriotic biopsy to ascertain the proteolytic activity response of TNF-α treatments.National Science Foundation (U.S.). Graduate Research FellowshipNational Institutes of Health (U.S.) (CDP Center Grant P50-GM68762)National Institutes of Health (U.S.) (Grant R01-GM081336)National Cancer Institute (U.S.) (Grant U54-CA112967)United States. Defense Advanced Research Projects Agency (Cipher Program