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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 (2019)

Chapter 1: Functionalization of nanocrystals is essential for their practical application, but synthesis on nanocrystal surfaces is limited by the available chemistries to introduce new functionality and create conjugated materials. Applications such as the development of imaging probes and hybrid materials require new methods for conjugating and interfacing nanoparticles with biological systems. The introductory chapter will present relevant background on the types of nanocrystals used in this work as well as the state and drawbacks of current nanocrystal functionalization techniques.

Chapter 2: As a basis for surface modification, the development of polymer encapsulation techniques was investigated to enable phase transfer to aqueous dispersions and to display reactive functional groups at the interface. Use of this encapsulation procedure allows for reproducible and tunable surface composition for a variety of chemistries and further modifications. Modifications can be used to display different functional groups on the nanoparticle surface for conjugation or to modulate physical properties.

Chapter 3: One surface conjugation strategy is the use of copper-catalyzed azide-alkyne cycloaddition (CuAAC). It is among the most popular methods for ligating molecules to surfaces, but as Cu(I) ions quickly and irreversibly quench semiconductor quantum dot (QD) fluorescence, development of this chemistry has been largely useless for QDs. A combinatorial fluorescence assay was developed to screen for non-quenching synthetic conditions for CuAAC on QD surfaces, and we identified conditions for complete coupling without significant quenching. Using these findings, I synthesized unquenched QD-peptidyl toxin conjugates and imaged their specific and voltage-dependent affinity for potassium channels in live cells.

Chapter 4: The use of the split domain SpyTag/SpyCatcher system forms the basis for another facet of conjugate design that can incorporate biomolecules through a small engineered peptide tag. This peptide-protein pair is a genetically-encodable tool to incorporate specific, covalent interactions between the two components which can be included in separate materials. The reaction is traceless and agnostic to a wide range of reaction conditions, integrating a degree of modularity into our systems. With this linkage as a design rule, we generate stable biomolecule conjugates as probes for imaging and as the basis for higher order structures.

Chapter 5: Self-assembly offers a scalable and reproducible bottom-up approach to fabricate patterned nanomaterials, but they have been limited in their ability to combine high conjugation yields with programmability. We employed protein engineering to modify the bacterial S-layer proteins SbsB and RsaA to create biomolecular scaffolds for the controlled deposition of multiple types of nanoparticles. Using the isopeptide bond-forming SpyCatcher conjugation, we have enabled dense coverage of a wide range of nanoparticles on the free-floating SbsB lattice including gold nanoparticles (Au NPs), quantum dots (QDs) and upconverting nanoparticles (UCNPs). Using orthogonal conjugation strategies, we created arrays with Au NP-QD pairings that conferred plasmonic enhancement of QD radiative decay. In addition to purified sheets, we show that SpyCatcher ligation of QDs to cell surface RsaA proteins produces crosslinking into an extended 3D cellular network. Confocal and atomic force microscopies demonstrate a dense and ordered layer of QDs on the cell surface, while mechanical analysis of the supracellular material demonstrates a >30-fold enhancement of storage modulus. The modularity inherent to the design tolerates changes to the nanocrystal composition and permits regeneration of the material after damage.

Chapter 6: While the applications of nanoparticle conjugates are varied, they all stem from the central need for methods that extend the reach of synthetic chemistry on the nanocrystal surface. Development of these techniques allows the creation of hybrid materials of one or more nanocrystal types interacting with proteinaceous and cellular biomaterials. I recapitulate these findings to describe the current state of the art and the future of the nanocrystal chemistry field.

    Cover page: Nanoparticle Conjugates and Materials through Manipulating Surface Chemistry
    • Article
    • Peer Reviewed
    LBL Publications (2021)
    Controlled patterning of nanoparticles on bioassemblies enables synthesis of complex materials for applications in optics, nanoelectronics, and sensing. Biomolecular self-assembly offers molecular control for engineering patterned nanomaterials, but current approaches have been limited in their ability to combine high nanoparticle coverage with generality that enables incorporation of multiple nanoparticle types. Here, we synthesize photonic materials on crystalline two-dimensional (2D) protein sheets using orthogonal bioconjugation reactions, organizing quantum dots (QDs), gold nanoparticles (AuNPs), and upconverting nanoparticles along the surface-layer (S-layer) protein SbsB from the extremophile Geobacillus stearothermophilus. We use electron and optical microscopy to show that isopeptide bond-forming SpyCatcher and SnoopCatcher systems enable the simultaneous and controlled conjugation of multiple types of nanoparticles (NPs) at high densities along the SbsB sheets. These NP conjugation reactions are orthogonal to each other and to Au-thiol bond formation, allowing tailorable nanoparticle combinations at sufficient labeling efficiencies to permit optical interactions between nanoparticles. Fluorescence lifetime imaging of SbsB sheets conjugated to QDs and AuNPs at distinct attachment sites shows spatially heterogeneous QD emission, with shorter radiative decays and brighter fluorescence arising from plasmonic enhancement at short interparticle distances. This specific, stable, and efficient conjugation of NPs to 2D protein sheets enables the exploration of interactions between pairs of nanoparticles at defined distances for the engineering of protein-based photonic nanomaterials.
      Cover page: Controlled and Stable Patterning of Diverse Inorganic Nanocrystals on Crystalline Two-Dimensional Protein Arrays
      • Article
      • Peer Reviewed
      UC Davis Previously Published Works (2018)
      Functionalization of nanocrystals is essential for their practical application, but synthesis on nanocrystal surfaces is limited by incompatibilities with certain key reagents. The copper-catalyzed azide-alkyne cycloaddition is among the most useful methods for ligating molecules to surfaces, but has been largely useless for semiconductor quantum dots (QDs) because Cu+ ions quickly and irreversibly quench QD fluorescence. To discover nonquenching synthetic conditions for Cu-catalyzed click reactions on QD surfaces, we developed a combinatorial fluorescence assay to screen >2000 reaction conditions to maximize cycloaddition efficiency while minimizing QD quenching. We identify conditions for complete coupling without significant quenching, which are compatible with common QD polymer surfaces and various azide/alkyne pairs. Based on insight from the combinatorial screen and mechanistic studies of Cu coordination and quenching, we find that superstoichiometric concentrations of Cu can promote full coupling if accompanied by ligands that selectively compete with the Cu from the QD surface but allow it to remain catalytically active. Applied to the conjugation of a K+ channel-specific peptidyl toxin to CdSe/ZnS QDs, we synthesize unquenched QD conjugates and image their specific and voltage-dependent affinity for K+ channels in live cells.
        Cover page: Azide–Alkyne Click Conjugation on Quantum Dots by Selective Copper Coordination
        • Article
        • Peer Reviewed
        LBL Publications (2020)
        Semiconductor quantum dots (QDs) have demonstrated utility in long-term single particle tracking of membrane proteins in live cells in culture. To extend the superior optical properties of QDs to more physiologically relevant cell platforms, such as acute brain slices, we examine the photophysics of compact ligand-conjugated CdSe/CdS QDs using both ensemble and single particle analysis in brain tissue media. We find that symmetric core passivation is critical for both photostability in oxygenated media and for prolonged single particle imaging in brain slices. We then demonstrate the utility of these QDs by imaging single dopamine transporters in acute brain slices, achieving 20 nm localization precision at 10 Hz frame rates. These findings detail design requirements needed for new QD probes in complex living environments, and open the door to physiologically relevant studies that capture the utility of QD probes in acute brain slices.
          Cover page: Ligand-conjugated quantum dots for fast sub-diffraction protein tracking in acute brain slices
          • Article
          • Peer Reviewed
          UC Berkeley Previously Published Works (2017)
          Semiconductor quantum dots (QDs) have proven to be superior probes for single-molecule imaging compared to organic or genetically encoded fluorophores, but they are limited by difficulties in protein targeting, their larger size, and on-off blinking. Here, we report compact aqueous CdSe/CdS QDs with significantly improved bioconjugation efficiency and superior single-molecule optical properties. We have synthesized covalent protein labeling ligands (i.e., SNAP tags) that are optimized for nanoparticle use, and QDs functionalized with these ligands label SNAP-tagged proteins ∼10-fold more efficiently than existing SNAP ligands. Single-molecule analysis of these QDs shows 99% of time spent in the fluorescent on-state, ∼4-fold higher quantum efficiency than standard CdSe/ZnS QDs, and 350 million photons detected before photobleaching. Bright signals of these QDs enable us to track the stepping movement of a kinesin motor in vitro, and the improved labeling efficiency enables tracking of single kinesins in live cells.
            Cover page: Covalent Protein Labeling and Improved Single-Molecule Optical Properties of Aqueous CdSe/CdS Quantum Dots
            • Article
            • Peer Reviewed
            LBL Publications (2019)
            Materials synthesized by organisms, such as bones and wood, combine the ability to self-repair with remarkable mechanical properties. This multifunctionality arises from the presence of living cells within the material and hierarchical assembly of different components across nanometer to micron scales. While creating engineered analogues of these natural materials is of growing interest, our ability to hierarchically order materials using living cells largely relies on engineered 1D protein filaments. Here, we lay the foundation for bottom-up assembly of engineered living material composites in 2D along the cell body using a synthetic biology approach. We engineer the paracrystalline surface-layer (S-layer) of Caulobacter crescentus to display SpyTag peptides that form irreversible isopeptide bonds to SpyCatcher-modified proteins, nanocrystals, and biopolymers on the extracellular surface. Using flow cytometry and confocal microscopy, we show that attachment of these materials to the cell surface is uniform, specific, and covalent, and its density can be controlled on the basis of the insertion location within the S-layer protein, RsaA. Moreover, we leverage the irreversible nature of this attachment to demonstrate via SDS-PAGE that the engineered S-layer can display a high density of materials, reaching 1 attachment site per 288 nm2. Finally, we show that ligation of quantum dots to the cell surface does not impair cell viability, and this composite material remains intact over a period of 2 weeks. Taken together, this work provides a platform for self-organization of soft and hard nanomaterials on a cell surface with precise control over 2D density, composition, and stability of the resulting composite, and is a key step toward building hierarchically ordered engineered living materials with emergent properties.
              Cover page: Engineering the S‑Layer of Caulobacter crescentus as a Foundation for Stable, High-Density, 2D Living Materials
              • Article
              • Peer Reviewed
              LBL Publications (2020)
              Semiconductor quantum dots (QDs) have demonstrated utility in long-term single particle tracking of membrane proteins in live cells in culture. To extend the superior optical properties of QDs to more physiologically relevant cell platforms, such as acute brain slices, we examine the photophysics of compact ligand-conjugated CdSe/CdS QDs using both ensemble and single particle analysis in brain tissue media. We find that symmetric core passivation is critical for both photostability in oxygenated media and for prolonged single particle imaging in brain slices. We then demonstrate the utility of these QDs by imaging single dopamine transporters in acute brain slices, achieving 20 nm localization precision at 10 Hz frame rates. These findings detail design requirements needed for new QD probes in complex living environments, and open the door to physiologically relevant studies that capture the utility of QD probes in acute brain slices.
              • 2 supplemental videos
              • 1 supplemental ZIP
              Cover page: Ligand-conjugated quantum dots for fast sub-diffraction protein tracking in acute brain slices.
              • Article
              • Peer Reviewed
              UC San Francisco Previously Published Works (2021)
              Inorganic nanocrystals such as quantum dots (QDs) and upconverting nanoparticles (UCNPs) are uniquely suited for quantitative live-cell imaging and are typically functionalized with ligands to study specific receptors or cellular targets. Antibodies (Ab) are among the most useful targeting reagents owing to their high affinities and specificities, but common nanocrystal labeling methods may orient Ab incorrectly, be reversible or denaturing, or lead to Ab-NP complexes too large for some applications. Here, we show that SpyCatcher proteins, which bind and spontaneously form covalent isopeptide bonds with cognate SpyTag peptides, can conjugate engineered Ab to nanoparticle surfaces with control over stability, orientation, and stoichiometry. Compact SpyCatcher-functionalized QDs and UCNPs may be labeled with short-chain variable fragment Ab (scFv) engineered to bind urokinase-type plasminogen activator receptors (uPAR) that are overexpressed in many human cancers. Confocal imaging of anti-uPAR scFv-QD conjugates shows the antibody mediates specific binding and internalization by breast cancer cells expressing uPAR. Time-lapse imaging of photostable scFv-UCNP conjugates shows that Ab binding causes uPAR internalization with a ∼20 min half-life on the cell surface, and uPAR is internalized to endolysosomal compartments distinct from general membrane stains and without significant recycling to the cell surface. The controlled and stable conjugation of engineered Ab to NPs enables targeting of diverse receptors for live-cell study of their distribution, trafficking, and physiology.
                Cover page: Immunotargeting of Nanocrystals by SpyCatcher Conjugation of Engineered AntibodiesCreative Commons 'BY-NC' version 4.0 license
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