Inferring diffusion in single live cells at the single molecule level

The movement of molecules inside living cells is a fundamental feature of biological processes. The ability to both observe and analyse the details of molecular diffusion in vivo at the single molecule and single cell level can add significant insight into understanding molecular architectures of diffusing molecules and the nanoscale environment in which the molecules diffuse. The tool of choice for monitoring dynamic molecular localization in live cells is fluorescence microscopy, especially so combining total internal reflection fluorescence (TIRF) with the use of fluorescent protein (FP) reporters in offering exceptional imaging contrast for dynamic processes in the cell membrane under relatively physiological conditions compared to competing single molecule techniques. There exist several different complex modes of diffusion, and discriminating these from each other is challenging at the molecular level due to underlying stochastic behaviour. Analysis is traditionally performed using mean square displacements of tracked particles, however, this generally requires more data points than is typical for single FP tracks due to photophysical instability. Presented here is a novel approach allowing robust Bayesian ranking of diffusion processes (BARD) to discriminate multiple complex modes probabilistically. It is a computational approach which biologists can use to understand single molecule features in live cells.Comment: combined ms (1-37 pages, 8 figures) and SI (38-55, 3 figures

Applications of AFM in pharmaceutical sciences

Atomic force microscopy (AFM) is a high-resolution imaging technique that uses a small probe (tip and cantilever) to provide topographical information on surfaces in air or in liquid media. By pushing the tip into the surface or by pulling it away, nanomechanical data such as compliance (stiffness, Young’s Modulus) or adhesion, respectively, may be obtained and can also be presented visually in the form of maps displayed alongside topography images. This chapter outlines the principles of operation of AFM, describing some of the important imaging modes and then focuses on the use of the technique for pharmaceutical research. Areas include tablet coating and dissolution, crystal growth and polymorphism, particles and fibres, nanomedicine, nanotoxicology, drug-protein and protein-protein interactions, live cells, bacterial biofilms and viruses. Specific examples include mapping of ligand-receptor binding on cell surfaces, studies of protein-protein interactions to provide kinetic information and the potential of AFM to be used as an early diagnostic tool for cancer and other diseases. Many of these reported investigations are from 2011-2014, both from the literature and a few selected studies from the authors’ laboratories

Array atomic force microscopy for real-time multiparametric analysis.

Nanoscale multipoint structure-function analysis is essential for deciphering the complexity of multiscale biological and physical systems. Atomic force microscopy (AFM) allows nanoscale structure-function imaging in various operating environments and can be integrated seamlessly with disparate probe-based sensing and manipulation technologies. Conventional AFMs only permit sequential single-point analysis; widespread adoption of array AFMs for simultaneous multipoint study is challenging owing to the intrinsic limitations of existing technological approaches. Here, we describe a prototype dispersive optics-based array AFM capable of simultaneously monitoring multiple probe-sample interactions. A single supercontinuum laser beam is utilized to spatially and spectrally map multiple cantilevers, to isolate and record beam deflection from individual cantilevers using distinct wavelength selection. This design provides a remarkably simplified yet effective solution to overcome the optical cross-talk while maintaining subnanometer sensitivity and compatibility with probe-based sensors. We demonstrate the versatility and robustness of our system on parallel multiparametric imaging at multiscale levels ranging from surface morphology to hydrophobicity and electric potential mapping in both air and liquid, mechanical wave propagation in polymeric films, and the dynamics of living cells. This multiparametric, multiscale approach provides opportunities for studying the emergent properties of atomic-scale mechanical and physicochemical interactions in a wide range of physical and biological networks

Plasmonic antennas and zero mode waveguides to enhance single molecule fluorescence detection and fluorescence correlation spectroscopy towards physiological concentrations

Single-molecule approaches to biology offer a powerful new vision to elucidate the mechanisms that underpin the functioning of living cells. However, conventional optical single molecule spectroscopy techniques such as F\"orster fluorescence resonance energy transfer (FRET) or fluorescence correlation spectroscopy (FCS) are limited by diffraction to the nanomolar concentration range, far below the physiological micromolar concentration range where most biological reaction occur. To breach the diffraction limit, zero mode waveguides and plasmonic antennas exploit the surface plasmon resonances to confine and enhance light down to the nanometre scale. The ability of plasmonics to achieve extreme light concentration unlocks an enormous potential to enhance fluorescence detection, FRET and FCS. Single molecule spectroscopy techniques greatly benefit from zero mode waveguides and plasmonic antennas to enter a new dimension of molecular concentration reaching physiological conditions. The application of nano-optics to biological problems with FRET and FCS is an emerging and exciting field, and is promising to reveal new insights on biological functions and dynamics.Comment: WIREs Nanomed Nanobiotechnol 201

Layer-By-Layer Assembly of Graphene Oxide on Thermosensitive Liposomes for Photo-Chemotherapy

Stimuli responsive polyelectrolyte nanoparticles have been developed for chemo-photothermal destruction of breast cancer cells. This novel system, called layer by layer Lipo-graph (LBL Lipo-graph), is composed of alternate layers of graphene oxide (GO) and graphene oxide conjugated poly (l-lysine) (GO-PLL) deposited on cationic liposomesencapsulating doxorubicin. Various concentrations of GO and GO-PLL were examined and the optimal LBL Lipo-graph was found to have a particle size of 267.9 ± 13 nm, zeta potentialof +43.9 ± 6.9 mV and encapsulation efficiency of 86.4 ± 4.7%. The morphology of LBL Lipo-graph was examined by cryogenic-transmission electron microscopy (Cryo-TEM), atomic force microcopy (AFM) and scanning electron microscopy (SEM). The buildup of LBL Lipo-graph was confirmed via ultraviolet-visible (UV–Vis) spectrophotometry, thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) analysis. Infra-red (IR) response suggests that four layers are sufficient to induce a gel-to-liquid phase transition in response to near infra-red (NIR) laser irradiation. Light-matter interaction of LBL Lipo-graph was studied by calculating the absorption cross section in the frequency domain by utilizing Fourier analysis. Drug release assay indicates that the LBL Lipo-graph releases much faster in an acidic environment than a liposome control. A cytotoxicity assay was conducted to prove the efficacy of LBL Lipo-graph to destroy MD-MB-231 cells in response to NIR laser emission. Also, image stream flow cytometry and two photon microcopy provide supportive data for the potential application of LBL Lipo-graph for photothermal therapy. Study results suggest the novel dual-sensitive nanoparticles allow intracellular doxorubin delivery and respond to either acidic environments or NIR excitation. Statement of Significance Stimuli sensitive hybrid nanoparticles have been synthesized using a layer-by-layer technique and demonstrated for dual chemo-photothermal destruction of breast cancer cells. The hybrid nanoparticles are composed of alternating layers of graphene oxide and graphene oxide conjugated poly-l-lysine coating the surface of a thermosensitive cationic liposome containing doxorubicin as a core. Data suggests that the hybrid nanoparticles may offer many advantages for chemo-photothermal therapy. Advantages include a decrease of the initial burst release which may result in the reduction in systemic toxicity, increase in pH responsivity around the tumor environment and improved NIR light absorption

Roadmap on semiconductor-cell biointerfaces.

This roadmap outlines the role semiconductor-based materials play in understanding the complex biophysical dynamics at multiple length scales, as well as the design and implementation of next-generation electronic, optoelectronic, and mechanical devices for biointerfaces. The roadmap emphasizes the advantages of semiconductor building blocks in interfacing, monitoring, and manipulating the activity of biological components, and discusses the possibility of using active semiconductor-cell interfaces for discovering new signaling processes in the biological world

Present and future of surface-enhanced Raman scattering

The discovery of the enhancement of Raman scattering by molecules adsorbed on nanostructured metal surfaces is a landmark in the history of spectroscopic and analytical techniques. Significant experimental and theoretical effort has been directed toward understanding the surface-enhanced Raman scattering (SERS) effect and demonstrating its potential in various types of ultrasensitive sensing applications in a wide variety of fields. In the 45 years since its discovery, SERS has blossomed into a rich area of research and technology, but additional efforts are still needed before it can be routinely used analytically and in commercial products. In this Review, prominent authors from around the world joined together to summarize the state of the art in understanding and using SERS and to predict what can be expected in the near future in terms of research, applications, and technological development. This Review is dedicated to SERS pioneer and our coauthor, the late Prof. Richard Van Duyne, whom we lost during the preparation of this article

Nanoscale integration of single cell biologics discovery processes using optofluidic manipulation and monitoring.

The new and rapid advancement in the complexity of biologics drug discovery has been driven by a deeper understanding of biological systems combined with innovative new therapeutic modalities, paving the way to breakthrough therapies for previously intractable diseases. These exciting times in biomedical innovation require the development of novel technologies to facilitate the sophisticated, multifaceted, high-paced workflows necessary to support modern large molecule drug discovery. A high-level aspiration is a true integration of "lab-on-a-chip" methods that vastly miniaturize cellulmical experiments could transform the speed, cost, and success of multiple workstreams in biologics development. Several microscale bioprocess technologies have been established that incrementally address these needs, yet each is inflexibly designed for a very specific process thus limiting an integrated holistic application. A more fully integrated nanoscale approach that incorporates manipulation, culture, analytics, and traceable digital record keeping of thousands of single cells in a relevant nanoenvironment would be a transformative technology capable of keeping pace with today's rapid and complex drug discovery demands. The recent advent of optical manipulation of cells using light-induced electrokinetics with micro- and nanoscale cell culture is poised to revolutionize both fundamental and applied biological research. In this review, we summarize the current state of the art for optical manipulation techniques and discuss emerging biological applications of this technology. In particular, we focus on promising prospects for drug discovery workflows, including antibody discovery, bioassay development, antibody engineering, and cell line development, which are enabled by the automation and industrialization of an integrated optoelectronic single-cell manipulation and culture platform. Continued development of such platforms will be well positioned to overcome many of the challenges currently associated with fragmented, low-throughput bioprocess workflows in biopharma and life science research