Nanomechanical bio-sensing for fast and reliable detection of viability and susceptibility of microorganisms

The metabolic activity of living organisms results in cellular motion on a nanometric scale that can be efficiently detected by micro- and nano- fabricated sensors. Quartz crystal microbalance and atomic force microscopy (AFM) inspired techniques have recently demonstrated their ability to successfully measure the nanometric motion of microorganisms. Monitoring these fluctuations while exposing the microorganisms to various compounds (e.g., metabolic inhibitors and drugs, fixatives, etc.) provides a rapid, label-free assessment of cellular activity and the live versus dead state of both prokaryotic and eukaryotic cells. To date, microbial activity-induced nanometric oscillations of AFM cantilevers have primarily been measured using commercially available AFMs. In this article we present a novel, user-friendly mechano-sensing device, termed a Nanomotion Detector (NMD), recently developed in our laboratories, that simplifies AFM systems. This NMD offers a streamlined design that is simple to align, is optimized for assays with live cells and liquid exchange, and can be operated in a Peltier-controlled incubator providing thermal control. Here, we successfully tested the ability of the NMD to discern differences between the live/dead nanometric motion of Escherichia coli and Staphlococcus aureus exposed to antibiotics and fixatives. This NMD is a dedicated cell activity detector that can advantageously replace commercially available AFMs for nanomotion detection applications including rapid antibiotic sensitivity testing.

In situ Studies of Peptide and Protein Assemblies at the Solid-Liquid Interface

Bart Willem Stel

At the interface between science and engineering, there is the field of biomimicry: Innovations inspired by the observation of natural, evolutionary optimized biological structures and processes. To understand the challenges of biomimicry, the concept of âMolecular Tectonicsâ is particularly useful. It describes how different self-assembling processes are spatially and dynamically integrated to form successively more complex structures. This integration makes it difficult to isolate the underlying mechanisms, which are often on the scale of individual molecules. Molecular biomimicry therefore critically depends on the observation of natural systems at the molecular scale. Advances in scanning probe microscopy have enabled the study of molecular self-assembly in unprecedented detail. However, many of these advances critically depend on the use of Ultra High Vacuum (UHV) conditions. To study natural systems at the molecular scale, a transition from UHV towards solvated systems is required. In the first part of this thesis, small synthetic peptides are used as model system for molecular self-assembly. The self-assembly of peptides at interfaces has been studied by systematically varying the chemical structure and deposition method. By using ElectroSpray Ion Beam Deposition (ES-IBD) as well as in situ drop casting, the self-assembly at the solid-vacuum interface (UHV conditions) and at the solid-liquid interface was directly compared. This comprehensive study on the effect of solvent, structure and deposition method on the assembly of peptides at gas-solid and solid-liquid biointerfaces has shown that the same AcFA5K peptide can organize into various structures, from Î²-sheets to tubular and globular assemblies, depending on their local environment. The study of molecular self-assembly was extended to 2D protein crystals. By using high-speed and high-resolution and Atomic Force Microscopy (AFM), it was possible to resolve both spatially and dynamically the mechanisms involved in S-layer self-assembly at the solid-liquid interface, such as monomer adsorption, condensation into high-density clusters, crystallization and finally conformational changes of existing S-layer crystals. In the second part of this thesis, the integration of different self-assembling systems is explored and their functional properties are investigated. It combines the concepts of self-assembly and molecular tectonics. Self-assembled striped PS-b-PEO block copolymer (BCP) thin film substrates were used to confine and align S-layer self-assembly, without affecting the internal structure. Furthermore, the nucleation rate and S-layer growth was greatly enhanced on PS-b-PEO substrates, compared to the pure PS or PEO constituents. The hierarchical use of different self-assembling systems can be seen as a functional approach to the concept of molecular tectonics. The catalytic properties of S-layers regarding the formation of CaCO3 were studied. By studying the catalytic properties of S-layers ex vivo, their role can be investigated independently from the internal metabolism of their parent bacteria. Synchrotron based in situ X-ray Absorption Spectroscopy (XAS) and AFM are combined with continuous flow conditions, demonstrating that S-layers are able to stabilize amorphous forms of CaCO3 and catalyze the nucleation of calcite at extremely low supersaturations.

EPFL2018

Atomic Force Microscopy of Biological Systems: Quantitative Imaging and Nanomotion Detection

Petar Stupar

The core of this thesis is the application of different modalities of atomic force microscopy to study living systems. After a brief introduction, quantitative imaging state of the art is presented and the applications for mammalian, plant, bacterial, and yeast cells are reviewed. The same chapter contains research effort about exploring nanomechanical properties of bone cells exposed to conditions that simulate the ones in outer space, in order to investigate the changes in architectural rearrangements of cells during space flight. The next section takes the focus on probing bacterial adhesion sites with an antibody-modified tip, a research important for the understanding of how bacterial cells adhere to the host and establish an infection. Plant cells and their nanomechanical profiles are also covered in the chapter. A large portion of the thesis consists in development of a new nanomechanical sensing technique, and the exploration of its potential applications. The technique is based on the AFM detection method and focuses on transducing small fluctuations that define living systems into a measurable mechanical change in the cantilever. The fact that there is a disproportionate rise in antimicrobial-resistant bacteria and that our lives depend on the new drugs and diagnostic tools to fight them has fueled the first and largest application of the technique â rapid antimicrobial susceptibility resting. Chapter 3 starts with the introduction of the technique, basic principles and guidelines for its use, and continues on potential applications. Proof of principle of the technique is presented, showing experimental results using blind clinical samples of bloodstream infectious agents. A separate section is devoted to investigations concerning slow-growing bacteria and the application of the technique to rapidly determine which antibiotic would best work against them. It then expands on the applications towards detecting metabolic activity of mitochondria and their response to substrates and inhibition. Furthermore, the method has been described as a rapid anti-cancer profiling tool that might pave the way towards a diagnostic platform for personalized medical treatment. The final section of the third chapter contains the current knowledge of the origins of oscillatory movement that governs the nanomotion technique. It presents different hypothesis and results towards exploring them. The thesis ends with a summary of the main ideas and conclusions about the presented work and future perspectives.

EPFL2018

Scaling properties of DNA knots studied by atomic force microscopy

Erika Ercolini

The main subject of the present thesis is the experimental study of the scaling properties of DNA knots of different complexities by tapping mode atomic force microscopy (AFM) in air. Homo- or heterogeneous mixture of DNA knot types were deposited onto mica in regimes of (i) strong adsorption, which induces a kinetic trapping of the molecules, and of (ii) weak adsorption, which permits relaxation on the surface. The contour of each knotted molecule was analyzed by a box counting algorithm, giving the number of boxes containing a part of the molecule, N(L), as a function of the box size, L, allowing to recover the relation N(L) ≈ L-df, where df is the fractal dimension and ν = 1/df is the scaling exponent. This relationship is complicated by the presence of a persistence length of DNA (about 50 nm) which introduces a crossover from a rigid rod behavior to a self-avoiding walk behavior. In (i) the radius of gyration of the adsorbed DNA knot scales with the 3D Flory exponent ν ≈ 0.58 within error. In (ii), the value ν ≈ 0.66, intermediate between the 3D and 2D (ν = 3/4) exponents, was found, indicating an incomplete 2D relaxation or a different polymer universality class. A different analysis, where the fractal dimension was determined by a customized box counting algorithm giving the knot mass as a function of the box size, yielded compatible results. In the case of weak binding conditions, AFM images show evidence of the localization of knot crossings, which is an effect theoretically predicted for knotted polymers confined in two dimensions (flat knots). Part of this thesis is dedicated to a fascinating application of AFM: the imaging of biomolecules in an aqueous buffer. In particular, images of plasmids adsorbed to mica strong enough to be visualized and loosely enough to see them moving in consecutive scans will be shown. The possibility of imaging under buffer molecules loosely anchored to the substrate opens the way to the investigation of biological processes near physiological conditions. The first step of the study of homologous recombination will be presented. We will also show images concerning our study of the activity of topoisomerase I on supercoiled plasmids. Clusters of DNA molecules and proteins were found when imaging in presence of magnesium, in agreement with recent findings by AFM in air. The research on topoisomerases and their interactions with inhibitors or poisons is a hot topic, being these enzymes targets of anti-cancer drugs. Our study paves the way to the investigation of the activity of topoisomerase II, the enzyme which removes knots from DNA. Finally, the principles of static and dynamic light scattering, and the results of dynamic light scattering on latex beads of known size will be presented. Particular emphasis will be given to the discussion of the contribution of light scattering techniques for future experiments on the study of the static and dynamic properties of DNA knotted molecules in solution. These experiments would contribute to shed light on the possible dependence of the gyration radius of knotted polymers on the knot type, and would allow testing theoretical predictions about their relaxation time.

EPFL2008

In situ Studies of Peptide and Protein Assemblies at the Solid-Liquid Interface

Bart Willem Stel

EPFL2018

Atomic Force Microscopy of Biological Systems: Quantitative Imaging and Nanomotion Detection

Petar Stupar

EPFL2018

Scaling properties of DNA knots studied by atomic force microscopy

Erika Ercolini

EPFL2008