OPTICAL DETECTION OF MOLECULAR INTERACTIONS ON THE SURFACE OF MATERIALS INDEX-MATCHED TO WATER.

The idea to have highly effective autonomous sensors able to measure and share information about the quality of our environment, and particularly water, in our lakes and rivers, our water supply system and the outputs of municipal and industrial wastewater treatment systems is revolutionary and fascinating. These sensors could be densely deployed at multiple locations, and the information may be available to citizens through the Internet. This idyllic vision, nowadays, is far away from being reality, despite the huge effort made to develop innovative molecular sensors. The main challenges related to the realization of these autonomous sensors network are the biofouling, power supply and compactness. In fact, despite thousands of papers in literature about development of novel nanostractured materials for sensing, for instance, there is still not a single example of any of these device being used in direct contact with water for long-term environmental monitoring. The work presented in this thesis proposes a new kind of optical sensor that combines a fast and low cost method to detect water pollutant with good performance and robustness. In particular, this work is focused on the detection of small molecular pollutants, as oils compounds and surfactants. An innovative aspect of the proposed approach relies on the use of a novel class of materials as sensing substrate which have peculiar and fascinating optical properties: these are amorphous perfluorinated polymers with refractive index similar to that of water. When immersed in aqueous solutions, they provide extremely low reflection or scattering of light, hence they become barely visible. For this reason, this class of materials is called phantom. In this limit, when a thin molecular layer spontaneously adsorbs on the surfaces of these materials, the reflected or scattered light increases, providing the basis for optical detection of molecules. In this work, three different phantom materials made of perfluorinated polymers are exploited in the framework of the detection of water contaminants: a prism, microporous membranes and micro-beads, that represent the building blocks for the assembly of an invisible chromatography column. The membrane and the micro-beads were produced for the first time during this work. The use of fluoropolymer prism substrate for molecular detection was already proposed in recent works to realize label-free biosensors based on the functionalization of the surface with antibodies. Here I extend the exploitation of this system to the detection of molecular pollutant through their adsorption on the bare surface of the fluoropolymer materials, without the need of any surface treatment. Despite the lack of surface functionalization, a selectivity in the adsorption of various classes of molecules is demonstrated

Laser-induced forward transfer (LIFT) of water soluble polyvinyl alcohol (PVA) polymers for use as support material for 3D-printed structures

The additive microfabrication method of laser-induced forward transfer (LIFT) permits the creation of functional microstructures with feature sizes down to below a micrometre [1]. Compared to other additive manufacturing techniques, LIFT can be used to deposit a broad range of materials in a contactless fashion. LIFT features the possibility of building out of plane features, but is currently limited to 2D or 2½D structures [2–4]. That is because printing of 3D structures requires sophisticated printing strategies, such as mechanical support structures and post-processing, as the material to be printed is in the liquid phase. Therefore, we propose the use of water-soluble materials as a support (and sacrificial) material, which can be easily removed after printing, by submerging the printed structure in water, without exposing the sample to more aggressive solvents or sintering treatments. Here, we present studies on LIFT printing of polyvinyl alcohol (PVA) polymer thin films via a picosecond pulsed laser source. Glass carriers are coated with a solution of PVA (donor) and brought into proximity to a receiver substrate (glass, silicon) once dried. Focussing of a laser pulse with a beam radius of 2 µm at the interface of carrier and donor leads to the ejection of a small volume of PVA that is being deposited on a receiver substrate. The effect of laser pulse fluence , donor film thickness and receiver material on the morphology (shape and size) of the deposits are studied. Adhesion of the deposits on the receiver is verified via deposition on various receiver materials and via a tape test. The solubility of PVA after laser irradiation is confirmed via dissolution in de-ionised water. In our study, the feasibility of the concept of printing PVA with the help of LIFT is demonstrated. The transfer process maintains the ability of water solubility of the deposits allowing the use as support material in LIFT printing of complex 3D structures. Future studies will investigate the compatibility (i.e. adhesion) of PVA with relevant donor materials, such as metals and functional polymers. References: [1] A. Piqué and P. Serra (2018) Laser Printing of Functional Materials. Weinheim, Germany: Wiley-VCH Verlag GmbH & Co. KGaA. [2] R. C. Y. Auyeung, H. Kim, A. J. Birnbaum, M. Zalalutdinov, S. A. Mathews, and A. Piqué (2009) Laser decal transfer of freestanding microcantilevers and microbridges, Appl. Phys. A, vol. 97, no. 3, pp. 513–519. [3] C. W. Visser, R. Pohl, C. Sun, G.-W. Römer, B. Huis in ‘t Veld, and D. Lohse (2015) Toward 3D Printing of Pure Metals by Laser-Induced Forward Transfer, Adv. Mater., vol. 27, no. 27, pp. 4087–4092. [4] J. Luo et al. (2017) Printing Functional 3D Microdevices by Laser-Induced Forward Transfer, Small, vol. 13, no. 9, p. 1602553

Optical feedback interferometry sensing technique for flow measurements in microchannels

Le phénomène d’interférométrie par réinjection optique, ou effet self-mixing dans un laser permet de concevoir des capteurs non-invasifs, auto-alignés, ne nécessitant que peu d’éléments optiques et simples à implémenter. Ce type de capteur permet de mesurer avec la précision propre à l’interférométrie laser le déplacement, la vitesse ou la position de cibles dite coopératives (cibles réfléchissantes ou fortement diffusantes). Dans cette étude, ce type de capteurs est appliqué à la mesure de profil d’écoulement des fluides dans des microcanaux. Le faible coût et la polyvalence des capteurs à réinjection optique sont d’un grand intérêt dans l’industrie biomédicale et chimique, ainsi que pour la recherche en mécanique des fluides. Dans un premier temps, et en se basant sur les études réalisées dans des macro-canaux, nous avons proposé un modèle d’interferométrie par réinjection optique dans une diode laser lorsque la cible est constitué de particules en mouvement, en suspension dans un liquide. A partir de ce modèle, nous avons étudié expérimentalement l’impact du volume de mesure ainsi que du type de particules (taille et concentration) sur le signal mesuré. Nous avons ensuite proposé des méthodes de traitement du signal permettant de calculer le calcul du débit du fluide, ainsi que sous certaines conditions identifiées, la vitesse locale en tout point d’un microcanal. Ces études préliminaires nous ont permis de reconstruire le profil d’écoulement de différents liquides dans des canaux de 320µm de diamètre. Enfin, nous avons comparé les performances du capteur développé dans cette thèse avec un capteur basé sur la technique du Dual-Slit, technique déjà validée pour la microfluidique, en mesurant le profil d’écoulement dans un canal à section rectangulaire de 100x20µm. ABSTRACT : The phenomenon of optical feedback interferometry (OFI) or self-mixing effect in a laser is used to design non-invasive and self-aligned sensors, requiring only few optical elements and simple to implement. This type of sensor is used to measure the displacement, velocity or position of cooperative targets (reflective or strongly scattering targets). In this study, this phenomenom is applied to the measurement of fluid flow profile in microchannels. The low cost and versatility of optical feedback sensors are of great interest in biomedical and chemical industry as well as research in fluid mechanics. Based on studies in macro-channels, we proposed first a theoretical model of OFI in a laser diode when the target consists of moving particles suspended in a liquid. Based on this model, we then studied experimentally the impact of the sensor’s sensing volume and the type of particles (size and concentration) on the OFI signal. We then proposed signal processing methods for calculating the fluid flow rate, as well as the local velocity at any point in a microchannel. These preliminary studies allowed us to reconstruct the flow profile of different liquids flowing in a circular channel of 320μm diameter. Finally, we compared the performance of the sensor developed in this thesis with a sensor based on the Dual-Slit technique, which has been already validated for microchannels, by measuring the flow profile in a rectangular shaped channel (100x20µm)

Single molecule detection and fluorescence correlation spectroscopy on surfaces

In this thesis a new approach for single molecule detection and analysis is explored. This approach is based on the combination of two well established methods, fluorescence correlation spectroscopy (FCS) and total internal reflection fluorescence microscopy (TIRFM). In contrast to most existing fluorescence spectroscopy techniques, the subject of primary interest in FCS is not the fluorescence intensity itself but the random intensity fluctuation around the mean value. Intensity fluctuations are induced by thermal noise in a minute observation volume, which is in classical FCS the confocal volume of a confocal microscope. E.g. FCS is commonly utilized to investigate diffusion. In this case, diffusing fluorescent molecules entering or leaving the observation volume cause intensity fluctuations, which are analyzed by calculating the temporal autocorrelation of the observed signal. The autocorrelation is a measure for the self-similarity of a signal and contains information about the average fluctuation strength and duration. The confocal observation volume, i.e. the measurement volume that is actually seen by the detector is approximately given by the product of the optical transfer function with the fluorescence exciting intensity distribution of a focused laser beam. To achieve a high signal-to-background ratio a small observation volume is absolutely essential, first of all because the background from e.g. scattered light increases with the size of the observation volume. Second, a small volume assures for a small average number of fluorophores inside the observation volume and therefore for a high fluctuation amplitude i.e. FCS signal. This thesis proposes and discusses an alternative to confocal FCS specially conceived for measurements on surface-bound molecular systems, such as biological receptors or immobilized enzymes. In contrast to confocal FCS, fluorescence is excited within an evanescent field generated by total internal reflection (TIR) of a laser beam at the interface between a microscope coverslip and the sample. This is achieved by focusing the laser beam off-axis at the back focal plane of a high NA oil-immersion objective. The collimated beam that emerges from the objective is incident at an oblique angle at the coverslip-sample interface and totally internal reflected. In contrast to confocal FCS, the generated observation volume is completely confined to the surface and background fluorescence as well as scattered light from the bulk is efficiently suppressed. Our method, called objective-type TIR-FCS in the following, features an increased collection efficiency compared to existing techniques that combine evanescent wave excitation and FCS. Existing techniques use total internal reflection on the surface of a prism to generate an evanescent field. This leads to a configuration where the choice of objectives is limited to air or water-immersion objectives. In our system we use a high NA oil-immersion objective, specially conceived for TIR applications, which collects light efficiently. The collection efficiency is further enhanced by a naturally occurring change of the emission properties of fluorophores close to interfaces between dielectric media. The presence of the interface favors emission into the optically denser medium so that about 60% of the emitted light can be collected. These factors, together with a reduced observation volume lead to a very sensitive method with a high potential for applications in single molecule detection and analysis. The performance of the proposed method was experimentally shown for measurements on molecules subject to Brownian motion and binding to modified coverslips. In particular, it was experimentally shown that objective-type TIR-FCS features high signal-to-background ratio on a single molecule level. In this thesis, concise derivations of analytical expressions for autocorrelation functions for diffusion and most important, the case of ligands reversibly binding to a single and localized binding site are presented. The derived model allows for the quantitative determination of binding rates for a single receptor. We strongly believe that the application of these results in the context of investigations of receptor-ligand binding kinetics will allow for deeper understanding of cellular signaling. Moreover, this thesis discusses the applicability of the proposed method in enzymology. Enzymes, as most proteins are subject to continuous changes of their structure or conformation. These conformational changes are correlated with the function of the enzyme. In the discussed example the enzyme catalyzes an oxidation where the product is fluorescent but the substrate is not. The function of the enzyme i.e. the recurring product formation leads to observable intensity fluctuations. Since function and conformational states are correlated, conformational fluctuations can be investigated by means of FCS as was already shown for confocal FCS. A technique closely related to FCS is fluorescence lifetime spectroscopy (where lifetime refers to the mean lifetime of the electronic excited state). Whereas in FCS the relaxation after random deviations from thermal equilibrium is investigated, relaxation of excited fluorophores towards their electronic ground-state is investigated in lifetime spectroscopy. The technique is used for e.g. discrimination between fluorophores with different lifetimes. Lifetime spectroscopy combined with imaging is used in many domains of life-science, including microarray reading and medical diagnostics. In preliminary work we developed a novel approach to perform lifetime imaging that is based on a multiplexing technique. The proposed method requires no mechanical scanning stage and only a single-point detector. Furthermore, noise is reduced under certain circumstances if the signal is low. Characteristics of this technique as well as advantages and disadvantages are shortly discussed

Advanced Image Acquisition, Processing Techniques and Applications

"Advanced Image Acquisition, Processing Techniques and Applications" is the first book of a series that provides image processing principles and practical software implementation on a broad range of applications. The book integrates material from leading researchers on Applied Digital Image Acquisition and Processing. An important feature of the book is its emphasis on software tools and scientific computing in order to enhance results and arrive at problem solution

Novel Biosensing Approaches for Detection of Exosomal Proteins

Exosomes are endocytic lipid membrane-bound bodies that have been shown to carry proteins associated with cancer and neurodegenerative disease. This has led to an exponential rise in research that looks to incorporate exosomal proteins as disease biomarkers within diagnostic assays. Furthermore, ubiquitous presence of exosomes in nearly all biological fluids creates the possibly of minimally invasive liquid biopsies for the patient. However, the heterogeneity of exosomes and complexity of biological source materials requires a consideration of optimal isolation protocols. More importantly, the development of effective exosome based assays is limited by the scarcity of translational characterization approaches that are capable of determining their molecular composition and physical properties in physiological fluids. The key objectives of this doctoral research was to establish a robust exosome isolation protocol from complex media, prior to sensing the exosomes on an immunosensor transduced by acoustic wave and electrochemical measurements. This work also looked to enhance these platforms from their current baseline performance, through the implementation of various surface structure modifications. A size exclusion chromatography approach was developed for the isolation of exosomes expressing CD63, Alix, CD81 and CD9 proteins, and allowed them to be extracted effectively from cell culture media, human serum and urine. Isolated exosomes were subsequently detected on a quartz crystal microbalance with dissipation (QCM-D) monitoring, after the optimisation of an affinity-based immunofunctionalisation approach. This technique displayed high sensitivity and specificity towards exosomal CD63 at clinically relevant concentrations in complex media. The QCM-D sensor was also used as a working electrode, as part of an electrochemical cell, to enable additional impedance spectroscopy analysis of exosome binding in tandem with the QCM-D response, collectively termed EQCM-D. The combination of these approaches offers a label-free, sensitive and real-time approach to exosome detection. The sensitivity of the EQCM-D platform was improved through surface formation of tuneable gold nanoparticle arrays from selective impregnation of block-copolymer templates, taking advantage of their segregation behaviour. This presented a versatile approach to tune sensor surfaces in order to improve ligand orientation and subsequent analyte binding. Similar advancements were made on silica detection surfaces through the formation of silica inverse opal crystals, with differing thicknesses, using a single-step co-assembly approach that combines a sol-gel matrix with poly(methyl methacrylate) (PMMA) spheres. Porous networks atop the sensors increased the internal surface area significantly, translating to a higher binding capacity of exosomes notwithstanding a higher degree of artefact entrapment. The results achieved through this work offer a potential for multi-modal analysis of exosomal proteins in diagnostics, underpinned by acoustic wave methodologies and nanostructured materials

Microelectromechanical Systems and Devices

The advances of microelectromechanical systems (MEMS) and devices have been instrumental in the demonstration of new devices and applications, and even in the creation of new fields of research and development: bioMEMS, actuators, microfluidic devices, RF and optical MEMS. Experience indicates a need for MEMS book covering these materials as well as the most important process steps in bulk micro-machining and modeling. We are very pleased to present this book that contains 18 chapters, written by the experts in the field of MEMS. These chapters are groups into four broad sections of BioMEMS Devices, MEMS characterization and micromachining, RF and Optical MEMS, and MEMS based Actuators. The book starts with the emerging field of bioMEMS, including MEMS coil for retinal prostheses, DNA extraction by micro/bio-fluidics devices and acoustic biosensors. MEMS characterization, micromachining, macromodels, RF and Optical MEMS switches are discussed in next sections. The book concludes with the emphasis on MEMS based actuators

A study of SNARE-mediated autophagosome clearance using fluorescence lifetime microscopy

Cell survival requires the turnover of toxic cellular material and recycling of biomolecules in low nutrient conditions. An efficient degradation system is therefore essential for disease prevention and its dysfunction has been linked to both neurodegeneration and oncogenesis. Bulk degradation is accomplished through the collection of cytoplasmic material in a unique sequestration vesicle, which forms de novo and subsequently deposits cargo in the lysosome for degradation. This process, known as autophagy, therefore requires membrane fusion between the autophagosomal vesicle and the lysosome. SNARE proteins mediate membrane fusion events and therefore their careful regulation ensures the proper organisation of the membrane trafficking network. The SNARE proteins governing autophagosome clearance have been identified as syntaxin 17, SNAP29 and VAMP8 and SNARE assembly appears to be positively regulated by VPS33A. This well established model of SNARE-mediated autophagosome clearance has not, however, been demonstrated within the spatiotemporal framework of the cell and little is known about how VPS33A modulates SNARE function. The research presented in this thesis therefore aims to determine the applicability of the proposed SNARE model within the cellular environment and to investigate the regulatory mechanisms controlling syntaxin 17 function. To accomplish this, carefully validated fluorescence colocalisation and time-resolved fluorescence lifetime imaging techniques were primarily employed. The limitations of these techniques were also considered for data interpretation and a novel prototype SPAD array technology, designed for high-speed time-correlated single photon counting, was trialled for widefield FLIM-FRET. FLIM-FRET revealed that VAMP8 has been incorrectly assigned as the dominant autophagosomal R-SNARE and VPS33A studies evidence a multi-modal regulation of Stx17 that diverges from other studied syntaxin family modulation mechanisms. A new model of SNAREmediated autophagosome clearance is therefore proposed, where syntaxin 17 engages with SNAP29 and VAMP7 to drive membrane fusion with the endolysosome in a manner governed by VPS33A and dependent on the phosphorylation status of syntaxin 17