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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 (2020)

Many diseases, such as neurodegenerative disorders, cancer, and autoimmune diseases are caused by or exhibit symptoms of abnormal regulation of signaling proteins. In order to obtain a molecular understanding of diseases, we require tools capable of probing the intricate signaling pathways that govern biological function. Understanding the role these proteins play as they circulate between cells will allow us to examine disease progression from an intercellular point of view. Currently, it is difficult to study these proteins in their natural environment, in vivo, because they are created and function on very broad time and length scales. Existing protein detection methods have several limitations as they are optimized for intracellular imaging, performed in vitro, require lengthy sample handling, function over short time scales, or have molecular recognition elements that are unstable in biological media.

This dissertation presents a modular platform to create optical nanosensors that are able to address current limitations in signaling protein detection. Nanosensor elements require optimization of both signal transduction and molecular recognition elements. Single-walled carbon nanotubes (SWCNTs) are nanoparticles that are ideal signal transducers for biological imaging. SWCNTs have optical properties well-suited for biological sensing such as infinite fluorescence lifetime, no blinking, small size, and fluorescence in the near-infrared region of the electromagnetic spectrum that is least attenuated by biological systems. Several SWCNT nanosensors have already been developed for signaling small molecule targets and peptides, but, to date, none have been created for signaling proteins.

In order to create a robust imaging platform based on SWCNTs for sensing signaling proteins, it is possible to couple molecular recognition elements to SWCNT signal transducers using dual noncovalent and covalent functionalization strategies. The development of noncovalent molecular recognition elements is explored using peptide mimetic polymers called peptoids. The discovery of a synthetic peptoid binding loop for wheat germ agglutinin protein as a proof-of-principle case study shows the ability to utilize diverse chemical materials to create a fully synthetic binding element for desired protein targets. Additionally, the development of covalently functionalized SWCNTs is explored for the creation of multifunctional optical SWCNT nanosensors. Here covalently functional groups provide functional handles that work synergistically with noncovalent passivation to enable the development of a diverse nanosensor toolbox.

The findings presented in this dissertation lay the foundation of valuable techniques and materials to optimize the binding to and study of signaling proteins. Future work to streamline the development of novel nanosensors is discussed, and the framework for the creation of other biological nanomaterial tools through these noncovalent and covalent techniques is also explored.

    Cover page: Development of Single-Walled Carbon Nanotube Protein Optical Nanosensors
    • Article
    • Peer Reviewed
    LBL Publications (2017)
    Surface engineering of nanoparticles has recently emerged as a promising technique for synthetic molecular recognition of biological analytes. In particular, the use of synthetic heteropolymers adsorbed onto the surface of a nanoparticle can yield selective detection of a molecular target. Synthetic molecular recognition has unique advantages in leveraging the photostability, versatility, and exceptional chemical stability of nanomaterials. In particular, single-walled carbon nanotubes (SWNT) exhibit a large Stokes shift and near infrared emission for maximum biological sample transparency. Optical biosensors with high signal transduction and molecular specificity can be synthesized with amphiphilic heteropolymers grafted to SWNT, and discovered by high-throughput screening. Herein, we describe the development and the characterization of surface-engineered nanoparticles, or synthetic antibodies, for protein detection.
      Cover page: Surface Engineering of Nanoparticles to Create Synthetic Antibodies.
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2020)
      Unpredictable and uncontrollable protein adsorption on nanoparticles remains a considerable challenge to achieving effective application of nanotechnologies within biological environments. Nevertheless, engineered nanoparticles offer unprecedented functionality and control in probing and altering biological systems. In this review, we highlight recent advances in harnessing the "protein corona" formed on nanoparticles as a handle to tune functional properties of the protein-nanoparticle complex. Towards this end, we first review nanoparticle properties that influence protein adsorption and design strategies to facilitate selective corona formation, with the corresponding characterization techniques. We next focus on literature detailing corona-mediated functionalities, including stealth to avoid recognition and sequestration while in circulation, targeting of predetermined in vivo locations, and controlled activation once localized to the intended biological compartment. We conclude with a discussion of biocompatibility outcomes for these protein-nanoparticle complexes applied in vivo. While formation of the nanoparticle-corona complex may impede our control over its use for the projected nanobiotechnology application, it concurrently presents an opportunity to create improved protein-nanoparticle architectures by exploiting natural or guiding selective protein adsorption to the nanoparticle surface.
        Cover page: Engineering at the nano-bio interface: harnessing the protein corona towards nanoparticle design and function
        • Article
        • Peer Reviewed
        LBL Publications (2019)
        Studying the single cell protein secretome offers the opportunity to understand how a phenotypically heterogeneous population of individual cells contribute to ensemble physiology and signaling. Polarized secretion events such as neurotransmitter release and cytokine signaling necessitates spatiotemporal information to elucidate structure-function relationships. Polymer functionalized single-walled carbon nanotube protein sensor arrays allow microscopic imaging of secreted protein footprints and enable the study of the spatiotemporal heterogeneity of protein secretion at the single-cell level. The protocols for carbon nanotube sensor creation, sensor array preparation, and imaging secreted proteins in both prokaryotic and mammalian cells are presented in this chapter. Secreted RAP1 and HIV-1 integrase proteins were used as proof-of-concept examples. Additionally, we discuss potential variety of protein and non-protein analyte effluxes that can be imaged using this platform, as well as current and future perspectives related to sensor development and deployment.
          Cover page: Synthetic probe development for measuring single or few-cell activity and efflux.
          • Article
          • Peer Reviewed
          UC Berkeley Previously Published Works (2018)
          Microscopic imaging of the brain continues to reveal details of its structure, connectivity, and function. To further improve our understanding of the emergent properties and functions of neural circuits, new methods are necessary to directly visualize the relationship between brain structure, neuron activity, and neurochemistry. Advances in engineering the chemical and optical properties of nanomaterials concurrent with developments in deep-tissue microscopy hold tremendous promise for overcoming the current challenges associated with in vivo brain imaging, particularly for imaging the brain through optically-dense brain tissue, skull, and scalp. To this end, developments in nanomaterials offer much promise toward implementing tunable chemical functionality for neurochemical targeting and sensing, and fluorescence stability for long-term imaging. In this review, we summarize current brain microscopy methods and describe the diverse classes of nanomaterials recently leveraged as contrast agents and functional probes for microscopic optical imaging of the brain.
            Cover page: Advances in nanomaterials for brain microscopy
            • Article
            • Peer Reviewed
            UC Berkeley Previously Published Works (2017)
            Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the optical window where biological samples are most transparent. Here, we outline techniques to adsorb amphiphilic polymers and polynucleic acids onto the surface of SWNTs to engineer their corona phases and create novel molecular sensors for small molecules and proteins. These functionalized SWNT sensors are both biocompatible and stable. Polymers are adsorbed onto the nanotube surface either by direct sonication of SWNTs and polymer or by suspending SWNTs using a surfactant followed by dialysis with polymer. The fluorescence emission, stability, and response of these sensors to target analytes are confirmed using absorbance and near-infrared fluorescence spectroscopy. Furthermore, we demonstrate surface immobilization of the sensors onto glass slides to enable single-molecule fluorescence microscopy to characterize polymer adsorption and analyte binding kinetics.
              Cover page: Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes.
              • Article
              • Peer Reviewed
              LBL Publications (2019)
              Genetic engineering of plants is at the core of sustainability efforts, natural product synthesis and crop engineering. The plant cell wall is a barrier that limits the ease and throughput of exogenous biomolecule delivery to plants. Current delivery methods either suffer from host-range limitations, low transformation efficiencies, tissue damage or unavoidable DNA integration into the host genome. Here, we demonstrate efficient diffusion-based biomolecule delivery into intact plants of several species with pristine and chemically functionalized high aspect ratio nanomaterials. Efficient DNA delivery and strong protein expression without transgene integration is accomplished in Nicotiana benthamiana (Nb), Eruca sativa (arugula), Triticum aestivum (wheat) and Gossypium hirsutum (cotton) leaves and arugula protoplasts. We find that nanomaterials not only facilitate biomolecule transport into plant cells but also protect polynucleotides from nuclease degradation. Our work provides a tool for species-independent and passive delivery of genetic material, without transgene integration, into plant cells for diverse biotechnology applications.
                Cover page: High aspect ratio nanomaterials enable delivery of functional genetic material without DNA integration in mature plants.
                • Article
                • Peer Reviewed
                UC Berkeley Previously Published Works (2017)
                A distinct advantage of nanosensor arrays is their ability to achieve ultralow detection limits in solution by proximity placement to an analyte. Here, we demonstrate label-free detection of individual proteins from Escherichia coli (bacteria) and Pichia pastoris (yeast) immobilized in a microfluidic chamber, measuring protein efflux from single organisms in real time. The array is fabricated using non-covalent conjugation of an aptamer-anchor polynucleotide sequence to near-infrared emissive single-walled carbon nanotubes, using a variable chemical spacer shown to optimize sensor response. Unlabelled RAP1 GTPase and HIV integrase proteins were selectively detected from various cell lines, via large near-infrared fluorescent turn-on responses. We show that the process of E. coli induction, protein synthesis and protein export is highly stochastic, yielding variability in protein secretion, with E. coli cells undergoing division under starved conditions producing 66% fewer secreted protein products than their non-dividing counterparts. We further demonstrate the detection of a unique protein product resulting from T7 bacteriophage infection of E. coli, illustrating that nanosensor arrays can enable real-time, single-cell analysis of a broad range of protein products from various cell types.
                  Cover page: Single-molecule detection of protein efflux from microorganisms using fluorescent single-walled carbon nanotube sensor arrays
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