Les bactéries sont les organismes les plus abondants dans le monde. Des études sur les bactéries peuvent être bénéfiques pour la recherche médicale, la qualité des ressources en eau et l'industrie alimentaire. La détection et le marquage fluorescent est une des méthodes les plus utilisées pour des objectifs bioanalytiques. Dans la recherche de marqueurs luminescents et stables, des nouvelles nanoparticules fluorescentes et auto-stabilisées à base de polymères (FNPs, 60 nm) et des chaînes de polymères fluorescents (FPCs, 5nm) ont été développées. Dans un premier chapitre, une méthodologie pour insérer ces FNPs dans la bactérie E.coli a été développée. Pour contrôler si les FNPs sont en effet internalisé, nous avons développé un protocole basé sur l'extinction de luminescence des FNPs par le bleu de méthylène. Dans un second chapitre, les biotines conjuguées de FNPs peuvent être utilisées pour étudier les protéines membranaires spécifiques. En utilisant un lien streptavidine-biotine, un "sandwich" est formé pour construire un pont entre des particules, des anticorps spécifiques et des bactéries. Les images de fluorescence SPR et les images SEM ont démontré l'interaction de la biotine conjuguée de FNPs avec la bactérie E.coli. Dans un troisième chapitre, les chaînes de polymères fluorescents de couleur verte (GFPCs) peuvent facilement entrer dans des bactéries E.coli. Les GFPCs peuvent marquer le cyctoplasme mais pas l'ADN. Les chaînes de polymères fluorescents de couleur rouge (RFPCs) peuvent marquer facilement et efficacement la membrane de bactérie E.coli. Les deux FPCs sont extrêmement brillantes et non toxiques, les chaînes sont solubles dans l'eau. Ce sont de nouveaux matériaux fluorescents pour le marquage interne et externe des bactéries. Dans le dernier chapitre, il est démontré que les FANPs sont sensibles au pH et peuvent être utilisées pour mesurer la croissance de la bactérie E.coli. Les nano-objets détectent rapidement et précisément la croissance des cellules. En effet, leur fluorescence est sensible au changement de pH résultant du métabolisme cellulaire. De plus, ces particules permettent une surveillance en continu d'un grand nombre d'échantillons pour des applications de criblage à haut débit. Les nanomatériaux présentés dans ce manuscrit sont des outils prometteurs pour les applications en biocapteurs et bioimagerie en raison de leur luminosité/brillance et photostabilité élevées ainsi que les possibilités de post-fonctionnalisation. / Bacteria are the most abundant organisms in the world. Investigations and studies on bacteria can be beneficial to medical research, water resources research and food industry. Fluorescent sensing and labeling are commonly used for bioanalytical purposes. In the quest for very bright and stable labels, novel polymer-based, self-stabilized, fluorescent nanoparticles (FNPs, 60 nm) and fluorescent polymer chains (FPCs, 5 nm) have been developed. In the first part, a methodology to insert these FNPs into E.coli bacteria was developed. To control if the FNPs are indeed internalized, we developed a protocol based upon FNPs luminescence quenching by methylene blue. In the second part, a "sandwich" system is built. By using a streptavidin-biotin link, a bridge between particles (FNP), specific antibodies and bacteria is built. SPR, fluorescent images and SEM images demonstrated the interaction of biotin conjugated FNPs with E.coli bacteria. In the third part, interactions of fluorescent polymer chains with bacteria are investigated. Green fluorescent polymer chains (GFPCs) can easily enter into E.coli bacteria. GFPCs can label the cytoplasm but not the DNA. Red fluorescent polymer chains (RFPCs) can label the membrane of E.coli bacteria easily and efficiently. Both FPCs are highly water-soluble, bright and non-toxic, they are novel fluorescent labels for internal and external biological labeling of bacteria. In the last part, it is demonstrated that pH sensitive FANPs can be used to measure the growth of E.coli. They detect rapidly and accurately bacterial growth by signaling the change of pH resulting from cellular metabolism. Moreover, these particles allow for continuous monitoring a large number of samples for high-throughput screening applications. The studied fluorescent nanomaterials are promising tools for biosensing and bioimaging applications due to their brightness, high photostability and rich functionalisation ability.

In the past decade, the field of biosensing has experienced an incredible pace of development, due to the compelling need for accurate and reliable tools for characterization of biomolecular kinetics. Specifically, label-free kinetic measurements are the most direct method for studying molecular binding, for example to establish the efficacy of drug-receptor interactions. For this reason, researchers in the pharmaceutical industry rely heavily on label-free detection for drug and antibody screening. Meanwhile, in the biosafety industry and healthcare, there is great demand for screening tools that can target biothreats, in order to accurately recognize the presence of toxins and pathogens with high sensitivity in diverse samples, such as bodily fluids, food and drinking water. This research topic has become particularly relevant during the recent pandemic, where vaccine development was carried out side by side with quantification and characterization of single viral particles. Here, we introduce a versatile biosensing platform capable of characterizing virtually any type of target compound, down to the single molecule level. For this work, we have improved the Interferometric Reflectance Imaging Sensor (IRIS) to perform accurate measurements of the binding kinetics of analytes ranging in molecular weight from less than 1kDa (small molecules) to more than 1MDa (biological nanoparticles). For the first time, we demonstrate multiplexed kinetic binding characterization of small molecules to surface immobilized antibody probes, as well as detection and phenotyping of large and complex analytes, on the same platform. The IRIS platform utilizes the optical interference signal produced by thinly layered substrates in order to precisely measure the thickness of a transparent film atop a silicon chip. In the context of this work, dynamic characterization of a wide range of biomolecular and nanoparticle targets was made possible by a multidimensional optimization, in order to improve both the sensitivity and the dynamic range of the instrument. Analysis of low molecular weight compounds required a significant increase in signal to noise ratio, which was achieved through averaging, as well as complete elimination of background solution effects ('bulk effect’). Additionally, the best surface chemistry for each application was identified by a new technique which consists of immobilizing capture probes on a multiplexed array of active polymers functionalized on the same sensor surface, allowing for simultaneous side-by-side comparison of their performance. Surface chemistry plays a huge role in kinetic measurements, in terms of probe functionality, steric hindrance, charge distribution and diffusion effects. Finally, imaging optics, illumination wavelength, and thickness of the silicon dioxide film were optimized to perform detection and phenotyping of large analytes, such as extracellular vesicles (EVs) and antibody-conjugated gold nanoparticles (mAb-GNPs). Results obtained from numerical simulations allowed for selection of the best experimental parameters for each application. Experimentally, mAb-GNPs were utilized to produce a real-time sandwich lateral flow assay. In this context, we demonstrated how the improved IRIS platform can bridge the gap between single-particle detection ('digital’ configuration) and bulk reflectance measurements ('analog’ configuration), creating a new 'hybrid' system (h-IRIS), which only requires minimal hardware adjustments to easily switch from one modality to the other. This brought a substantial improvement in sensitivity, improving the limit of detection by three orders of magnitude and enabling single-molecule level measurements. Finally, future system optimization ideas are presented to achieve even higher accuracy and further extend the range of target analytes.

While 3D printing technologies first appeared in the 1980s, prohibitive costs, limited materials, and the relatively small number of commercially available printers confined applications mainly to prototyping for manufacturing purposes. As technologies, printer cost, materials, and accessibility continue to improve, 3D printing has found widespread implementation in research and development in many disciplines due to ease-of-use and relatively fast design-to-object workflow. Several 3D printing techniques have been used to prepare devices such as milli- and microfluidic flow cells for analyses of cells and biomolecules as well as interfaces that enable bioanalytical measurements using cellphones. This review focuses on preparation and applications of 3D-printed bioanalytical devices.

Clear plastic fluidic devices with ports for incorporating electrodes to enable electrochemiluminescence (ECL) measurements were prepared using a low-cost, desktop three-dimensional (3D) printer based on stereolithography. Electrodes consisted of 0.5 mm pencil graphite rods and 0.5 mm silver wires inserted into commercially available 1/4 in.-28 threaded fittings. A bioimaging system equipped with a CCD camera was used to measure ECL generated at electrodes and small arrays using 0.2 M phosphate buffer solutions containing tris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrate ([Ru(bpy)3]2+) with 100 mM tri-n-propylamine (TPA) as the coreactant. ECL signals produced at pencil graphite working electrodes were linear with respect to [Ru(bpy)3]2+ concentration for 9-900 μM [Ru(bpy)3]2+. The detection limit was found to be 7 μM using the CCD camera with exposure time set at 10 s. Electrode-to-electrode ECL signals varied by ±7.5%. Device performance was further evaluated using pencil graphite electrodes coated with multilayer poly(diallyldimethylammonium chloride) (PDDA)/DNA films. In these experiments, ECL resulted from the reaction of [Ru(bpy)3]3+ with guanines of DNA. ECL produced at these thin-film electrodes was linear with respect to [Ru(bpy)3]2+ concentration from 180 to 800 μM. These studies provide the first demonstration of ECL measurements obtained using a 3D-printed closed-channel fluidic device platform. The affordable, high-resolution 3D printer used in these studies enables easy, fast, and adaptable prototyping of fluidic devices capable of incorporating electrodes for measuring ECL.

The adrenal medulla is the inner part of adrenal glands located above each kidney, that produces catecholamines. Neuroblastoma and pheochromocytoma are the most prevalent malignancies of the adrenal medulla. Quantitative diagnosis of urinary catecholamines using HPLC-coupled Mass detectors is the current method for the diagnosis of neuroblastoma and pheochromocytoma. There are two major problems with this approach, (i) Because the catecholamines concentrations have short half-life (10-100 s), a series of urine tests must be performed throughout 24hr, detecting each catecholamine separately, is inconvenient and time-consuming; (ii) mass detectors are expensive, bulky, and require highly skilled personal. Vanillylmandelic (VMA), and homavanillic acid (HVA) are the by-products of catecholamines and are emerging alternative biomarker for catecholamines due to their high stability. Here, we developed a rapid, sensitive, miniaturized, and cheaper sensing platform for simultaneous quantifications of dopamine (DA), VMA, and HVA, with the aid of iron single-atom catalysts (Fe-SACs), based electrochemical sensor. SACs are atomically distributed metal atoms that have a maximum atomic utility rate of nearly 100%, compared to 30% for traditional metal nanoparticles. MXene sheets are employed to stabilize Fe-SACs, where, the exposed lone pairs of MXene serve as sites covalently linking high-energy single Fe atoms. MXene/Fe-SACs were synthesized by treating Ti3C2TxMXene with Iron chloride via freeze-drying followed by annealing. The successful formation of the material was verified by state-of-the-art characterizations. The MXene/Fe-SACs show superior electrocatalytic performance to the commonly used Fe- nanomaterials. Then, it was coated on the electrode surface and used to analyze DA, VMA, and HVA simultaneously via cyclic voltammetry (CV) and square-wave voltammetry (SWV). Under optimized conditions, the MXene/Fe-SACs electrochemical sensor showed detection limits as low as 1 nM and a linear range between 1 nM-100 μM for DA, LOD of 5 nM & linear range of 10 nM-100 μM VMA, and LOD of 10 nM & linear range of 20 nM-100 μM HAV. The method proved successful in detecting biomarkers in (spiked) synthetic urine and human serum. Furthermore, the method was successfully demonstrated in the determination of DA release from PC12 live cells, suggesting the wide practical use of SACs in sensing catecholamines-related metabolites.

Oxytocin (OXT), a neuropeptide synthesized in the paraventricular nucleus (PVN) of the hypothalamus, functions to increase the precedence of social stimuli and promote the development of a wide range of social behaviors. However, whether OXT has a predicting role in social reward has yet to be examined. In this study, we developed a genetically encoded, scalable OXT sensor named OXTR-iTango2 and applied this technique to define the role of OXT in learned social behaviors. OXTR-iTango2 enables the combination of light- and ligand- dependent gene expression both in vitro and in vivo neural systems. In order to study the predictive role of OXT during expected socially rewarding experiences, we first conditioned animals to a social environment, and then selectively labeled OXT-sensitive ventral tegmental area dopamine (VTA-DA) neurons when animals encountered a conditioned stimulus that stood to predict a familiar social reward. Recurrent exposure to the same social stimulus normally lowered the degree of social interaction, but this reduced interaction was not observed when OXT-sensitive DA neurons were optogenetically inhibited. Thus, our findings support the notion that OXT plays a role beyond promoting social interactions, leading for a new proposed hypothesis that OXT mediation also leads to active avoidance of mundane social interactions. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2020. / FAU Electronic Theses and Dissertations Collection

The microcantilever has become a ubiquitous tool for surface science, chemical sensing, biosensing, imaging, and energy harvesting, among many others. It is a device of relatively simple geometry with a static and dynamic response that is well understood. Further, because of it's small size, it is extremely sensitive to small external perturbations. These characteristics make the microcantilever an ideal candidate for a multitude of sensing applications. In this thesis dissertation we use the microcantilever to conduct numerous physical measurements and to study fundamental phenomena in the areas of fluid dynamics, turbulence, and biology. In each area we use the cantilever as a sensitive transducer in order to probe fluctuating forces. In micro and nanometer scale flows the characteristic length scale of the flow approaches and is even exceeded by the fluid mean free path. This limit is beyond the applicability of the Navier-Stokes equations, requiring a rigorous treatment using kinetic theory. In our first study, we conduct a series of experiments in which we use a microcantilever to measure gas dissipation in a nanoscopically confined system. Here, the distance between the gas molecules is of the same order as the separation between the cantilever and the walls of its container. As the cantilever is brought towards the wall, the flow becomes confined in the gap between the cantilever and the wall, affecting the resonant frequency and dissipation of the cantilever. By carefully tuning the separation distance, the gas pressure, and the cantilever oscillation frequency, we study the flow over a broad range of dimensionless parameters. Using these measurements, we provide an in-depth characterization of confinement effects in oscillating nanoflows. In addition, we propose a scaling function which describes the flow in the entire parameter space and which unifies previous theories based on the slip boundary condition and effective viscosity. In our next study, we seek to gain a better understanding of the transition to turbulence in a channel flow. We use a cantilever embedded in the channel wall to perform two sets of experiments: first, we study transition to turbulence triggered by the natural imperfections of the channel walls and second, we study transition under artificially added inlet noise. Our results point to two very different paths to turbulence. In the first case, wall effects lead to an extremely intermittent transitional flow and in the second case, broadband fluctuations originating at the inlet lead to less intermittent flow that is more reminiscent of homogeneous turbulence. The two experiments result in random flows in which high-order moments of near-wall fluctuations differ by orders of magnitude. Surprisingly however, the lowest order statistics in both cases appear qualitatively similar and can be described by a proposed noisy Landau equation. The noise, regardless of its origin, regularizes the Landau singularity of the relaxation time and makes transitions driven by different noise sources appear similar. Our results provide evidence of the existence of a finite turbulent relaxation time in transitional flows due to the persistent nature of noise in the system. In our last study, we turn to biologically-driven fluctuations from bacterial motion. Recent studies suggest that the motion of living bacteria could serve as a good indicator of bacteria species and resistance to antibiotics. To gain a better understanding of these fluctuations, we measure the nanomechanical motion of bacteria adhered to a chemically functionalized silicon microcantilever. A non-specific binding agent is used to attach E. coli to the surface of the device. The motion of the bacteria couples efficiently to the cantilever well below its resonance frequency, causing a measurable increase in its mechanical fluctuations. We vary the bacterial concentration over two orders of magnitude and are able to observe a corresponding change in the amplitude of fluctuations. Additionally, we administer antibiotics (Streptomycin) to kill the bacteria and observe a decrease in the fluctuations. A basic physical model is used to explain the observed spectral distribution of the mechanical fluctuations. These results lay the groundwork for understanding the motion of microorganisms adhered to surfaces and for developing micromechanical sensors for rapid bacterial identification and antibiotic resistance testing.

Au cours de ma thèse, j'ai développé un procédé de fabrication spécifique capable de produire un biocapteur qui combine deux techniques de biodétection différentes, la réponse plasmonique basée sur la résonance de plasmon de surface localisée (LSPR) et la réponse électrochimique. Les méthodes et les résultats qui sont présentés dans ce manuscrit ont été définis pour converger vers un dispositif fluidique unique combinant ces deux approches de détection différentes. Afin de trouver la configuration permettant l'excitation des résonances plasmoniques, la géométrie des nanocavités MIM (métal/isolant/métal) en réseau de lignes interdigitées a été optimisée par des simulations électromagnétiques. La fabrication par nanoimpression douce assistée UV (SoftUV-NIL) a été optimisée et, finalement, la caractérisation optique de ces nanocavités a été comparée avec succès aux simulations théoriques. Parallèlement à la réalisation de ce dispositif nanostructuré, des dispositifs électrochimiques fluidiques plus simples qui intègrent des microélectrodes classiques ont également été développés. L'objectif était d'abord de développer une chimie innovante pour le couple « biotine/streptavidine » et de comprendre ensuite comment les paramètres fluidiques peuvent affecter l'efficacité de capture des biomolécules. Ce manuscrit se termine par une discussion sur le rôle des paramètres fluidiques concernant l’efficacité de la biodétection sur la base de la théorie de Squires. / During my thesis, I worked on the development of a specific fabrication process able to produce a device that combines two different biodetection techniques, plasmonic response based on Localized Surface Plasmon Resonance (LSPR) and electrochemical response. Methods and results that are presented in this manuscript were defined to converge towards a unique fluidic device combining these two different sensing approaches. This device integrates interdigitated array of MIM nanocavities. In order to find the easier working configuration allowing the excitation of plasmonic resonances, their geometry has been optimized through electromagnetic simulations. The fabrication of these dual devices has been optimized based on Soft-UV NIL and, finally, optical characterization of these nanocavities has been successfully compared with theoretical simulations. In parallel to this challenging goal, simpler fluidic electrochemical devices that integrate conventional microelectrodes have also been developed. The goal was first to develop an innovative chemistry for the couple biotin/streptavidin and secondly to learn how fluidic parameters can affect the capture efficiency of molecules. This manuscript ends with a discussion on the role of the fluidic parameters on the biodetection efficiency based on the theory of Squires.

abstract: My research centers on the design and fabrication of biomolecule-sensing devices that combine top-down and bottom-up fabrication processes and leverage the unique advantages of each approach. This allows for the scalable creation of devices with critical dimensions and surface properties that are tailored to target molecules at the nanoscale. My first project focuses on a new strategy for preparing solid-state nanopore sensors for DNA sequencing. Challenges for existing nanopore approaches include specificity of detection, controllability of translocation, and scalability of fabrication. In a new solid-state pore architecture, top-down fabrication of an initial electrode gap embedded in a sealed nanochannel is followed by feedback-controlled electrochemical deposition of metal to shrink the gap and define the nanopore size. The resulting structure allows for the use of an electric field to control the motion of DNA through the pore and the direct detection of a tunnel current through a DNA molecule. My second project focuses on top-down fabrication strategies for a fixed nanogap device to explore the electronic conductance of proteins. Here, a metal-insulator-metal junction can be fabricated with top-down fabrication techniques, and the subsequent electrode surfaces can be chemically modified with molecules that bind strongly to a target protein. When proteins bind to molecules on either side of the dielectric gap, a molecular junction is formed with observed conductances on the order of nanosiemens. These devices can be used in applications such as DNA sequencing or to gain insight into fundamental questions such as the mechanism of electron transport in proteins. / Dissertation/Thesis / Doctoral Dissertation Physics 2019

Many drugs and environmental chemicals, such as antibiotics and chemotherapeutic agents, are nephrotoxic (toxic to the kidney) and are a common cause of acute kidney injury and chronic kidney disease. Conventional tissue models for assessment of drug-induced nephrotoxicity rely on animals or simple cell culture models, which lack tissue characteristics of the human kidney required to accurately predict a drug’s effect in clinical trials. Microfluidic kidney-on-chips can generate tissue with improved human relevance compared to traditional models, however, generally lack high-throughput and multiparametric data collection capabilities for evaluation of nephrotoxic drug exposures. Standard data collection techniques remain limited to fluorescent imaging or colorimetric assays that often focus on single endpoints, are invasive due to the addition of labels, and fail to capture dynamic changes in tissue function. Additionally, conventional toxicological readouts rely on bulk measures of injury, such as cell death, which are less sensitive than sub-lethal changes in cell function and morphology that occur prior to cell death. Due to the challenges above, there is a need for new measurement approaches that enable collection of kinetic, multi-parametric, and sub-lethal readouts of injury in kidney-on-chip systems. In this work, we developed and characterized several measurement approaches for evaluation of tissue function in kidney-on-chip systems and assessment of drug-induced nephrotoxicity. In chapter 2, we developed a novel optical-based oxygen sensing technique for measurement of sub-lethal mitochondrial dysfunction in an array of kidney-on-chips. In chapter 3, we investigated an approach for simultaneous transepithelial electrical resistance (TEER) sensing and flow control to enable near-continuous monitoring of tissue barrier function under different flow conditions. In chapter 4, we demonstrated the use of different data collection modalities, including multiple sensors, fluorescent imaging, and colorimetric-based assays, to generate multi-parametric readouts for evaluation of drug-induced nephrotoxicity in kidney-on-chips. / 2024-05-24T00:00:00Z