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Open Access Publications from the University of California

Scholarly Works (5 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2017)

Selective measurement of proteins from single cells is an important and challenging task in the study of cell biology. Protein signaling underlies many important cellular processes such as stem cell differentiation and tumor metastasis. However, due to the diversity of proteins and the subtlety of the chemical modifications that transduce the signals, it is challenging to selectively measure a specific protein from single cells. Further, determining the localization of proteins in individual cells is challenging due to the artifacts introduced by the chemical fixation required by current tools. To address each of these analytical gaps, we introduce two new single cell protein analysis platforms and two supporting technologies.

First, to eliminate fixation-induced artifacts in single-cell protein localization measurements, we developed the subcellular western blotting platform. We designed a set of buffers that enabled independent separations of the cytoplasmic and nuclear compartments of the same single cell. To enable buffer exchange without disturbing the intact cellular components, we developed a hydrogel-based lid for the delivery of lysis reagents purely by diffusion. We characterized the selectivity of the fractionation by assaying a panel of proteins with known localization and demonstrated the platform with an NF-$\kappa$B translocation assay.

Next, to enhance the selectivity of single-cell protein analysis, introduced an assay for performing single-cell isoelectric focusing. Building upon the hydrogel lid based reagent delivery system, we developed a microfluidic device that integrates sample preparation, analyte separation, and detection. We characterized the theoretical performance of the device using a fluorescent ladder and purified proteins. Further, we used the single-cell isoelectric focusing assay to separate isoforms and TurboGFP and $\beta$-tubulin from single cells. Additionally, we extended the platform by designing a lid system that generates a rectangular array of pH gradients, increasing the potential throughput of the single-cell isoelectric focusing assay.

To enable the rapid analysis of arrayed microscale separation data, we developed a suite of image processing scripts. We wrote the scripts in a modular manner both reducing complexity and enabling facile development of new functionality. We user-tested the scripts with life science researchers with backgrounds ranging from statistics to biology. From the user feedback, we identified the quality control step to be the most susceptible to user-to-user variance. Thus, on-going work aims to develop algorithms to automate the quality control process.

Finally, we report a method to prototype microscale separation devices. Polydimethylsiloxane is a common material used for rapid prototyping of microfludic devices. However, because it is oxygen permeable, it is not suitable for polyacrylamide gel electrophoresis since oxygen inhibits free radical polymerization. Thus, we developed a method to prevent oxygen inhibition and thus enable polymerization of polyacrylamide in PDMS microchannels. We characterized the separation performance of the resulting polyacrylamide hydrogels and demonstrated separation in reversibly sealed devices.

In summary, in this dissertation, we used our understanding of mass transport and microfabrication to design new platforms for selective single-cell protein analysis measurement. Future innovation may extend the sensitivity and multiplexing of these tools to enable highly specific measurement of all the key proteins in entire cellular signaling pathways.

    Cover page: Microfluidic tools for single-cell protein analysis
    • Article
    • Peer Reviewed
    UC Berkeley Previously Published Works (2016)
    To measure protein isoforms in individual mammalian cells, we report single-cell resolution isoelectric focusing (scIEF) and high-selectivity immunoprobing. Microfluidic design and photoactivatable materials establish the tunable pH gradients required by IEF and precisely control the transport and handling of each 17-pL cell lysate during analysis. The scIEF assay resolves protein isoforms with resolution down to a single-charge unit, including both endogenous cytoplasmic and nuclear proteins from individual mammalian cells.
      Cover page: Detection of Isoforms Differing by a Single Charge Unit in Individual Cells
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2016)
      This protocol describes how to perform western blotting on individual cells to measure cell-to-cell variation in protein expression levels and protein state. Like conventional western blotting, single-cell western blotting (scWB) is particularly useful for protein targets that lack selective antibodies (e.g., isoforms) and in cases in which background signal from intact cells is confounding. scWB is performed on a microdevice that comprises an array of microwells molded in a thin layer of a polyacrylamide gel (PAG). The gel layer functions as both a molecular sieving matrix during PAGE and a blotting scaffold during immunoprobing. scWB involves five main stages: (i) gravity settling of cells into microwells; (ii) chemical lysis of cells in each microwell; (iii) PAGE of each single-cell lysate; (iv) exposure of the gel to UV light to blot (immobilize) proteins to the gel matrix; and (v) in-gel immunoprobing of immobilized proteins. Multiplexing can be achieved by probing with antibody cocktails and using antibody stripping/reprobing techniques, enabling detection of 10+ proteins in each cell. We also describe microdevice fabrication for both uniform and pore-gradient microgels. To extend in-gel immunoprobing to gels of small pore size, we describe an optional gel de-cross-linking protocol for more effective introduction of antibodies into the gel layer. Once the microdevice has been fabricated, the assay can be completed in 4-6 h by microfluidic novices and it generates high-selectivity, multiplexed data from single cells. The technique is relevant when direct measurement of proteins in single cells is needed, with applications spanning the fundamental biosciences to applied biomedicine.
        Cover page: Single cell–resolution western blotting
        • Article
        • Peer Reviewed
        UC San Francisco Previously Published Works (2020)
        Comprehensive understanding of the serological response to SARS-CoV-2 infection is important for both pathophysiologic insight and diagnostic development. Here, we generate a pan-human coronavirus programmable phage display assay to perform proteome-wide profiling of coronavirus antigens enriched by 98 COVID-19 patient sera. Next, we use ReScan, a method to efficiently sequester phage expressing the most immunogenic peptides and print them onto paper-based microarrays using acoustic liquid handling, which isolates and identifies nine candidate antigens, eight of which are derived from the two proteins used for SARS-CoV-2 serologic assays: spike and nucleocapsid proteins. After deployment in a high-throughput assay amenable to clinical lab settings, these antigens show improved specificity over a whole protein panel. This proof-of-concept study demonstrates that ReScan will have broad applicability for other emerging infectious diseases or autoimmune diseases that lack a valid biomarker, enabling a seamless pipeline from antigen discovery to diagnostic using one recombinant protein source.
          Cover page: ReScan, a Multiplex Diagnostic Pipeline, Pans Human Sera for SARS-CoV-2 Antigens
          • Article
          • Peer Reviewed
          UCLA Previously Published Works (2020)
          Ageing is characterized by a progressive loss of physiological integrity, leading to impaired function and increased vulnerability to death1. Despite rapid advances over recent years, many of the molecular and cellular processes that underlie the progressive loss of healthy physiology are poorly understood2. To gain a better insight into these processes, here we generate a single-cell transcriptomic atlas across the lifespan of Mus musculus that includes data from 23 tissues and organs. We found cell-specific changes occurring across multiple cell types and organs, as well as age-related changes in the cellular composition of different organs. Using single-cell transcriptomic data, we assessed cell-type-specific manifestations of different hallmarks of ageing-such as senescence3, genomic instability4 and changes in the immune system2. This transcriptomic atlas-which we denote Tabula Muris Senis, or 'Mouse Ageing Cell Atlas'-provides molecular information about how the most important hallmarks of ageing are reflected in a broad range of tissues and cell types.
            Cover page: A single-cell transcriptomic atlas characterizes ageing tissues in the mouse
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