Ultrasound delivery of Surface Enhanced InfraRed Absorption active gold-nanoprobes into fibroblast cells: a biological study via Synchrotron-based InfraRed microanalysis at single cell level

Ultrasound (US) induced transient membrane permeabilisation has emerged as a hugely promising tool for the delivery of exogenous vectors through the cytoplasmic membrane, paving the way to the design of novel anticancer strategies by targeting functional nanomaterials to specific biological sites. An essential step towards this end is the detailed recognition of suitably marked nanoparticles in sonoporated cells and the investigation of the potential related biological effects. By taking advantage of Synchrotron Radiation fourier transform infrared micro-spectroscopy (SR-microftiR) in providing highly sensitive analysis at the single cell level, we studied the internalisation of a nanoprobe within fibroblasts (NIH-3T3) promoted by low-intensity US. To this aim we employed 20 nm gold nanoparticles conjugated with the IR marker 4-aminothiophenol. The significant Surface Enhanced Infrared Absorption provided by the nanoprobes, with an absorbance increase up to two orders of magnitude, allowed us to efficiently recognise their inclusion within cells. Notably, the selective and stable SR- microftiR detection from single cells that have internalised the nanoprobe exhibited clear changes in both shape and intensity of the spectral profile, highlighting the occurrence of biological effects. Flow cytometry, immunofluorescence and murine cytokinesis-block micronucleus assays confirmed the presence of slight but significant cytotoxic and genotoxic events associated with the US-nanoprobe combined treatments. our results can provide novel hints towards US and nanomedicine combined strategies for cell spectral imaging as well as drug delivery-based therapies

Modeling and Control of Piezoactive Micro and Nano Systems

Piezoelectrically-driven (piezoactive) systems such as nanopositioning platforms, scanning probe microscopes, and nanomechanical cantilever probes are advantageous devices enabling molecular-level imaging, manipulation, and characterization in disciplines ranging from materials science to physics and biology. Such emerging applications require precise modeling, control and manipulation of objects, components and subsystems ranging in sizes from few nanometers to micrometers. This dissertation presents a comprehensive modeling and control framework for piezoactive micro and nano systems utilized in various applications. The development of a precise memory-based hysteresis model for feedforward tracking as well as a Lyapunov-based robust-adaptive controller for feedback tracking control of nanopositioning stages are presented first. Although hysteresis is the most degrading factor in feedforward control, it can be effectively compensated through a robust feedback control design. Moreover, an adaptive controller can enhance the performance of closed-loop system that suffers from parametric uncertainties at high-frequency operations. Comparisons with the widely-used PID controller demonstrate the effectiveness of the proposed controller in tracking of high-frequency trajectories. The proposed controller is then implemented in a laser-free Atomic Force Microscopy (AFM) setup for high-speed and low-cost imaging of surfaces with micrometer and nanometer scale variations. It is demonstrated that the developed AFM is able to produce high-quality images at scanning frequencies up to 30 Hz, where a PID controller is unable to present acceptable results. To improve the control performance of piezoactive nanopositioning stages in tracking of time-varying trajectories with frequent stepped discontinuities, which is a common problem in SPM systems, a supervisory switching controller is designed and integrated with the proposed robust adaptive controller. The controller switches between two control modes, one mode tuned for stepped trajectory tracking and the other one tuned for continuous trajectory tracking. Switching conditions and compatibility conditions of the control inputs in switching instances are derived and analyzed. Experimental implementation of the proposed switching controller indicates significant improvements of control performance in tracking of time-varying discontinuous trajectories for which single-mode controllers yield undesirable results. Distributed-parameters modeling and control of rod-type solid-state actuators are then studied to enable accurate tracking control of piezoactive positioning systems in a wide frequency range including several resonant frequencies of system. Using the extended Hamilton\u27s principle, system partial differential equation of motion and its boundary conditions are derived. Standard vibration analysis techniques are utilized to formulate the truncated finite-mode state-space representation of the system. A new state-space controller is then proposed for asymptotic output tracking control of system. Integration of an optimal state-observer and a Lyapunov-based robust controller are presented and discussed to improve the practicability of the proposed framework. Simulation results demonstrate that distributed-parameters modeling and control is inevitable if ultra-high bandwidth tracking is desired. The last part of the dissertation, discusses new developments in modeling and system identification of piezoelectrically-driven Active Probes as advantageous nanomechanical cantilevers in various applications including tapping mode AFM and biomass sensors. Due to the discontinuous cross-section of Active Probes, a general framework is developed and presented for multiple-mode vibration analysis of system. Application in the precise pico-gram scale mass detection is then presented using frequency-shift method. This approach can benefit the characterization of DNA solutions or other biological species for medical applications

Roughness, wetting, and optical properties of functional surfaces

Funktionale Oberflächen mit einstellbaren Benetzungseigenschaften sind von enormem Interesse für hochwertige optische Komponenten sowie für Massenprodukte mit ästhetischen Anforderungen (z.B. easy-to-clean Brillengläser, Fenster oder beschlagfreie Visiere und Badezimmerspiegel). Gegenstand der vorliegenden Arbeit war die Entwicklung einer Mess- und Auswertemethodologie zur komplexen Charakterisierung der Struktur-Eigenschaftsbeziehung hydrophober und hydrophiler Funktionsflächen bis hin zur Superhydrophobie und zu Anti-Beschlageffekten. Dazu wurden bestehende Verfahren der Rauheits- und Benetzungsanalyse hinsichtlich ihrer Eignung für Benetzungssysteme mit unterschiedlich stochastisch rauen Oberflächen und intrinsischen Materialeigenschaften ausgewählt, angepasst und um neu eingeführte Methoden erweitert

Roughness, wetting, and optical properties of functional surfaces

Funktionale Oberflächen mit einstellbaren Benetzungseigenschaften sind von enormem Interesse für hochwertige optische Komponenten sowie für Massenprodukte mit ästhetischen Anforderungen (z.B. easy-to-clean Brillengläser, Fenster oder beschlagfreie Visiere und Badezimmerspiegel). Gegenstand der vorliegenden Arbeit war die Entwicklung einer Mess- und Auswertemethodologie zur komplexen Charakterisierung der Struktur-Eigenschaftsbeziehung hydrophober und hydrophiler Funktionsflächen bis hin zur Superhydrophobie und zu Anti-Beschlageffekten. Dazu wurden bestehende Verfahren der Rauheits- und Benetzungsanalyse hinsichtlich ihrer Eignung für Benetzungssysteme mit unterschiedlich stochastisch rauen Oberflächen und intrinsischen Materialeigenschaften ausgewählt, angepasst und um neu eingeführte Methoden erweitert

Towards conductive textiles: coating polymeric fibres with graphene

This is the final version of the article. Available from Springer Nature via the DOI in this record.Conducting fibres are essential to the development of e-textiles. We demonstrate a method to make common insulating textile fibres conductive, by coating them with graphene. The resulting fibres display sheet resistance values as low as 600 Ωsq−1, demonstrating that the high conductivity of graphene is not lost when transferred to textile fibres. An extensive microscopic study of the surface of graphene-coated fibres is presented. We show that this method can be employed to textile fibres of different materials, sizes and shapes, and to different types of graphene. These graphene-based conductive fibres can be used as a platform to build integrated electronic devices directly in textiles.The authors would like to thank Dr Yat-Tarng (Tommy) Shyng for the non-contact scanning measurements and would like to acknowledge financial support from the UK Engineering and Physical Sciences Research Council (EPSRC grants EP/J000396/1, EP/K017160, EP/K010050/1, EP/G036101/1, EP/M002438/1, EP/M001024/1), the Royal Society Travel Exchange Grants 2012 and 2013, the European Commission FP7-ICT-2013-613024-GRASP and H2020-MSCA-IF-2015-704963, and the Portuguese Foundation for Science and Technology (FCT), co-financed by FEDER (PT2020 Partnership Agreement), under contracts PTDC/QEQ-SUP/1413/2012, RECI/CTM-CER/0336/2012, IF/01088/2014, BI/UI89/2015, POCI-01-0145-FEDER-007679 (UID/CTM/50011/2013) and COMPETE:FCOMP-01-0124-FEDER-027465

Simultaneous Fluorescence and Atomic Force Microscopy to study Mechanically-Induced Bacterial Death in Real Time

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Física de la Materia Condensada. Fecha de lectura: 18-09-2020In the last decades, advanced imaging techniques have improved our ability to analyze biological systems at the micro and nanoscale, and in real time. Microscopy techniques have their own strengths and limitations, so their combination has the potential to provide a more comprehensive understanding of biological processes. This thesis is focused on the development and application of simultaneous fluorescence and atomic force microscopy (AFM) to study mechanically-induced bacterial death. The results reported here provide a quantitative understanding of the mechanical interactions between the AFM tip and bacteria, in the context of emerging mechano-bactericidal nanomaterials. This manuscript is divided into six chapters and one appendix. Chapter 1 provides an overview of the bacterial world and the strategies used over the years to combat the increasing bacterial contamination of surfaces, emphasizing the recent strategy based on mechanical damage. It also describes the microscopy techniques used, highlighting the strengths and weaknesses of each one, and discussing why correlative microscopy is more suitable to study this kind of processes. Chapter 2 describes the general materials and methods applied in this thesis and the software used to analyze experimental data. Chapter 3 provides the groundwork to develop a methodology to successfully combine AFM nanoindentation and fluorescence microscopy simultaneously using fluorescent polymer beads, focusing on the challenges that may arise when simultaneous measurements are performed. In Chapter 4, the methodology was adapted to image bacteria in physiological conditions, and optimal protocols to perform reproducible experiments on living bacteria were found. This optimized methodology in combination with a fluorescent cell membrane integrity marker was successfully applied to quantify the forces needed to rupture the bacterial cell wall. Moreover, a correlation between the forces exerted on bacteria and the kinetics of the fluorescence response is found. Chapter 4 is complemented by Appendix A, which provides the mechanical characterization of the bacterial wall below the rupture point, in order to give a more complete overview of the mechanical properties of the bacterial surface. Chapter 5 explores a different method to assess bacterial viability upon nanoindentation by monitoring the oscillation of the Min system, which reflects bacterial physiology. This method reveals that forces below the breakage point of the cell wall produce a fatigue effect, and provides a quantitative framework to understand low force collisions between bacteria and nanomaterials. These experiments also emphasize the limitation of integrity markers to provide a comprehensive view of bacterial response. The aim of Chapter 6 is to provide coherence and perspective to the main results of the thesis, as well as an outlook on how advanced microscopy methods and future experiments may impact the study of interactions between bacteria and nanotopographical features in the context of mechano-bactericidal nanomaterial