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Open Access Publications from the University of California

Scholarly Works (8 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2015)

Seminal bioanalytical technologies for high-throughput analysis, such as flow cytometry and capillary electrophoresis, were leveraging microfluidic physical phenomena long before the advent of the term “microfluidics”. Transitioning from the initial solid-state micro-electronic fabrication approaches, microfluidic fabrication has moved towards polymer based technologies that are amenable to a rapid design, prototype, and test development cycle. In my dissertation, I took advantage of these features to create new tools for performing electrophoresis-based protein assays over a range of applications, including, rapid low-power electrophoretic immunoassays, open-microfluidic ‘soft-MEMS’ platform for high-throughput protein analysis, and spatially & temporally controlled separation media for enhanced single-cell western blotting assays.

Rapid low-power electrophoretic immunoassay: To reduce the power requirements for a portable electrophoresis platform, we developed a new assay format that minimized the separation length to 1 mm for an electrophoretic immunoassay. The polyacrylamide gel moving boundary electrophoresis (PAGMBE) assay consumed just 3 μW and was completed in less than 30 seconds using only a 9 V battery - the lowest voltage reported for an electrophoretic protein separation.

Open-microfluidic ‘soft-MEMS’ platform: We developed an open-channel hydrogel architecture for rapid protein analysis. Directly photo-patterned free-standing polyacrylamide gel (fsPAG) microstructures support electrophoretic performance rivalling that of microfluidic platforms while maintaining easy interfacing with automated robotic controllers and downstream processing (e.g., sample spotters, immunological probing). We demonstrated 96 concurrent SDS protein fsPAGE separations in under 5 minutes.

Spatially varied separation media: Grayscale mask photo-patterning of gel density realized periodic 1 mm gradient gels arrays for single cell analysis. These dense arrays with a spatially varied sieving medium enabled concurrent protein sizing for 1000s of single cells in parallel. We demonstrated the single cell gradient gel western blotting to analyze multiple targets in the Her2 signaling pathway in a primary breast cancer tissue that varied by an order of magnitude in molecular weight. This broad-range protein sizing 1 mm gradient gel condition corresponds to the molecular weight range for 78% of the human proteome.

Pore-expansion for enhanced intra-gel assays: Protein separations in dense sieving media followed by in-gel immobilization enable high performance separations and minimal protein loss. This assay integration is necessary for processing low abundance samples – for example the protein content of a single cell. Unfortunately, for dense sieving medium prevents easy access for downstream assays on the immobilized proteins. We developed an active hydrogel matrix that could maintain a dense form to achieve a high-resolution protein separation and immobilization, then after a simulation, could transition to a lower-density form that is more accessible to antibody probing and the delivery of other large-reagents.

In summary, this dissertation focused on high-throughput protein analysis platforms that are designed towards the goal of massively parallelized proteomics, a major unrealized goal from bioanalytical technology.

    Cover page: Microfluidic Bioanalytical Device and Assay Development for High-Throughput Electrophoretic Protein Analysis
    • Article
    • Peer Reviewed
    UC Berkeley Previously Published Works (2016)
    High throughput, efficient, and readily adoptable analytical tools for the validation and selection of reliable antibody reagents would impact the life sciences, clinical chemistry, and clinical medicine. To directly quantify antibody-antigen association and dissociation rate constants, kon and koff, in a single experiment, we introduce a microfluidic free-standing kinetic polyacrylamide gel electrophoresis (fsKPAGE) assay. Here, an antibody is immobilized in zones along the length of a single freestanding polyacrylamide gel lane of varying cross-sectional width. Fluorescently labeled antigen is electrophoresed through each immobilized antibody zone, with local cross-sectional area determining the local electric field strength and, thus, the local interaction time between immobilized antibody and electromigrating antigen. Upon crossing, the interaction yields immobilized immunocomplex. The kon is quantified by assessing the amount of immunocomplex formed at each interaction time. To quantify koff, immobilized zones of fluorescently labeled immunocomplex are subjected to a buffer dilution and monitored over time. We determine kon and koff for prostate-specific antigen (PSA) and make a comparison to gold-standard values. The fsKPAGE assay determines kon and koff in a single experiment of less than 20 min, using 45 ng of often limited antibody material and standard laboratory equipment. We see the fsKPAGE assay as forming the basis for rapid, quantitative antibody-screening tools.
      Cover page: Kinetic Rate Determination via Electrophoresis along a Varying Cross-Section Microchannel
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2015)
      The underlying physical properties of microfluidic tools have led to new biological insights through the development of microsystems that can manipulate, mimic and measure biology at a resolution that has not been possible with macroscale tools. Microsystems readily handle sub-microlitre volumes, precisely route predictable laminar fluid flows and match both perturbations and measurements to the length scales and timescales of biological systems. The advent of fabrication techniques that do not require highly specialized engineering facilities is fuelling the broad dissemination of microfluidic systems and their adaptation to specific biological questions. We describe how our understanding of molecular and cell biology is being and will continue to be advanced by precision microfluidic approaches and posit that microfluidic tools - in conjunction with advanced imaging, bioinformatics and molecular biology approaches - will transform biology into a precision science.
        Cover page: Microfluidics: reframing biological enquiry
        • Article
        • Peer Reviewed
        LBL Publications (2018)
        Surface-assisted laser desorption ionization (SALDI) is an approach for gas-phase ion generation for mass spectrometry using laser excitation on typically conductive or semiconductive nanostructures. Here, we introduce insulator nanostructure desorption ionization mass spectrometry (INDI-MS), a nanostructured polymer substrate for SALDI-MS analysis of small molecules and peptides. INDI-MS surfaces are produced through the self-assembly of a perfluoroalkyl silsesquioxane nanostructures in a single chemical vapor deposition silanization-step. We find that surfaces formed from the perfluorooctyltrichlorosilane monomer assemble semielliptical features with a 10 nm height, diameters between 10 and 50 nm, and have attomole-femtomole sensitivities for selected analytes. Surfaces prepared with silanes that either lack the trichloro or perfluoro groups, lack sensitivity. Further, we demonstrate that hydrophobic INDI regions can be micropatterned onto hydrophilic surfaces to perform on-chip self-desalting in an array format.
          • 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 Berkeley Previously Published Works (2014)
            We describe a platform for high-throughput electrophoretic mobility shift assays (EMSAs) for identification and characterization of molecular binding reactions. A photopatterned free-standing polyacrylamide gel array comprised of 8 mm-scale polyacrylamide gel strips acts as a chassis for 96 concurrent EMSAs. The high-throughput EMSAs was employed to assess binding of the Vc2 cyclic-di-GMP riboswitch to its ligand. In optimizing the riboswitch EMSAs on the free-standing polyacrylamide gel array, three design considerations were made: minimizing sample injection dispersion, mitigating evaporation from the open free-standing polyacrylamide gel structures during electrophoresis, and controlling unit-to-unit variation across the large-format free-standing polyacrylamide gel array. Optimized electrophoretic mobility shift conditions allowed for 10% difference in mobility shift baseline resolution within 3 min. The powerful 96-plex EMSAs increased the throughput to ∼10 data/min, notably more efficient than either conventional slab EMSAs (∼0.01 data/min) or even microchannel based microfluidic EMSAs (∼0.3 data/min). The free-standing polyacrylamide gel EMSAs yielded reliable quantification of molecular binding and associated mobility shifts for a riboswitch-ligand interaction, thus demonstrating a screening assay platform suitable for riboswitches and potentially a wide range of RNA and other macromolecular targets.
              Cover page: High-Throughput Electrophoretic Mobility Shift Assays for Quantitative Analysis of Molecular Binding Reactions
              • Article
              • Peer Reviewed
              UC Berkeley Previously Published Works (2016)
              Pore-gradient microgel arrays enable thousands of parallel high-resolution single-cell protein electrophoresis separations for targets accross a wide molecular mass (25-289 kDa), yet within 1 mm separation distances. Dual crosslinked hydrogels facilitate gel-pore expansion after electrophoresis for efficient and uniform immunoprobing. The photopatterned, light-activated, and acid-expandable hydrogel underpins single-cell protein analysis, here for oncoprotein-related signaling in human breast biopsy.
                Cover page: Hydrogel Pore‐Size Modulation for Enhanced Single‐Cell Western Blotting
                • Article
                • Peer Reviewed
                UC Berkeley Previously Published Works (2018)
                Beneficial plant-microbe interactions offer a sustainable biological solution with the potential to boost low-input food and bioenergy production. A better mechanistic understanding of these complex plant-microbe interactions will be crucial to improving plant production as well as performing basic ecological studies investigating plant-soil-microbe interactions. Here, a detailed description for ecosystem fabrication is presented, using widely available 3D printing technologies, to create controlled laboratory habitats (EcoFABs) for mechanistic studies of plant-microbe interactions within specific environmental conditions. Two sizes of EcoFABs are described that are suited for the investigation of microbial interactions with various plant species, including Arabidopsis thaliana, Brachypodium distachyon, and Panicum virgatum. These flow-through devices allow for controlled manipulation and sampling of root microbiomes, root chemistry as well as imaging of root morphology and microbial localization. This protocol includes the details for maintaining sterile conditions inside EcoFABs and mounting independent LED light systems onto EcoFABs. Detailed methods for addition of different forms of media, including soils, sand, and liquid growth media coupled to the characterization of these systems using imaging and metabolomics are described. Together, these systems enable dynamic and detailed investigation of plant and plant-microbial consortia including the manipulation of microbiome composition (including mutants), the monitoring of plant growth, root morphology, exudate composition, and microbial localization under controlled environmental conditions. We anticipate that these detailed protocols will serve as an important starting point for other researchers, ideally helping create standardized experimental systems for investigating plant-microbe interactions.
                  Cover page: Ecosystem Fabrication (EcoFAB) Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions
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