Electrochemiluminescence (ECL) is a phenomenon whereby electrochemical reactions generate a product that is capable of emitting light. ECL’s high sensitivity, selectivity, extremely low background, and relatively simple instrumentation make it particularly well-suited for chemical sensing and biosensing strategies. Here we report a simple ECL imaging system based on a camera interfaced with a zoom lens to compare pencil graphite electrode (PGE) and screen-printed carbon electrode (SPCE) arrays as ECL platforms. With this system, ECL signals generated from tris(2,2′- bipyridine)ruthenium(II) chloride hexahydrate ([Ru(bpy)3]2+) using co-reactant tri-n-propylamine (TPA) were linear with respect to [Ru(bpy)3]2+ concentrations from 9 to 450 μM. Detection limits for [Ru(bpy)3]2+ were found to be 1.8 μM with PGEs and 0.9 μM with SPCEs. Immobilization of a thin polyvinylpyridine (PVP) film ECL reporter [Ru(bpy)2(PVP)10]2+ on SPCEs was also investigated. Overall, the combination of PGEs or SPCEs with the simple ECL imaging system offers a cost-effective approach to ECL-based sensing and biosensing.

No description available.

No description available.

There is a need for the development of new technologies for the early detection of disease. Diverse initiatives are underway in academia and the pharmaceutical and biotechnology industries to develop highly-sensitive, high-throughput methods to detect disease-relevant biomarkers at the single-cell level. Biomarkers can define the progress of a disease or efficacy of disease treatment, and can include nucleic acids (RNA, DNA), proteins, small molecules, or even specific cells. While discovery research in this area is accelerating, there are a number of current experimental limitations. Most existing methodologies require a relatively large sample size. Also, amplification-based detection technologies are destructive to sample, and errors in amplification can occur, leading to an incorrect diagnosis. Nanomaterial-based devices (nanodevices) offer the promise of label-free, amplification-free detection strategies. Such nanodevices could allow analysis of minute biological samples without the requirement for amplification or incorporation of reporter groups. Loss of sample, due to handling and processing would be minimized and the sample could be recovered for further analysis. Atomic force microscopy (AFM) allows topographic imaging and compressibility/elasticity measurement of biomolecules on solid supports. AFM can enable assays of ligand binding with single molecule detection capability. Certain nucleic acid types, in particular double-stranded (ds) RNA, can act as a biomarker for specific cancers (e.g. leukemia) and viral infection. dsRNA also is of interest since it is a conserved structural feature of precursors to gene-regulatory RNAs, including micro (mi) RNAs and short interfering (si) RNAs. This project demonstrates a single-step, label-free, amplification-free approach for detecting the interaction of biomolecules that bind and/or process dsRNA, using a nanomanipulated, self-assembled monolayer (SAM) of a ds[RNA-DNA] chimera as imprinting matrix, a reference nuclease as imprinting agent, and AFM for imprint-readout. The action of the dsRNA-specific enzyme, ribonuclease III (RNase III), as well as the binding of an inactive, dsRNA-binding RNase III mutant can be permanently recorded by the input-responsive action of a restriction endonuclease that cleaves an ancillary reporter site within the dsDNA segment. The resulting irreversible height change of the arrayed ds[RNA-DNA] chimera, as measured by atomic force microscopy, provides a distinct digital output for each type of input. These findings provide the basis for developing imprinting-based nano-biosensors, and reveal the versatility of AFM as a tool for characterizing the behaviour of highly-crowded biomolecules at the nanoscale. RNA-DNA heteroduplexes are biomarkers for specific inflammatory conditions of genetic origin, and also are the product of capture of an RNA (e.g., miRNA) by a complementary DNA sequence. The approach used here to detect RNA-DNA hybrids is based on the ability of alkylthiol-modified ssDNA molecules to form monolayers and nanomatrices on gold surfaces (as described above) with density-dependent thickness, which increases upon formation of RNA-DNA hybrids following addition of a complementary oligoribonucleotide. Changes in hybrid matrix thickness can be measured by AFM, using a reference monolayer. RNA-DNA hybrid formation as well as subsequent processing by RNase H can be observed as a height increase or decrease, respectively, of the monolayer. When Mg2+ is omitted to prevent RNA cleavage, but not protein binding, a significant height increase is observed. The height increase is not observed with the corresponding ssDNA or ssRNA nanomatrices, and only occurs with nanomatrices having a hybrid density above a defined threshold. The data indicate formation of a stable multimeric RNase H assembly on the hybrid nanomatrix which provides a robust signal that is nondestructive to the RNA. The implications of these findings are discussed with respect to development of novel detection methodologies for RNA, dsRNA, and RNA-DNA hybrids. / Chemistry

Development of repellent surfaces which can supress bacteria adhesion, blood contamination and thrombosis, and non-specific adhesion on diagnostic devices has been a topic of intense research as these characteristics are in high demand. This thesis focused on design and development of omniphobic surfaces based on hierarchical structures and their application for preventing pathogenic contamination and biosensing. First, a flexible hierarchical heat-shrinkable wrap featuring micro and nanostructures, was developed with straightforward scalable methods which can be applied to existing surfaces. These surfaces reduced biofilm formation of World Health Organization-designated priority pathogens as well as minimized risk of spreading contamination from intermediate surfaces. This is due to the broad liquid repellency and the presence of reduced anchor points for bacterial adhesion on the hierarchical surfaces. Next, the developed surfaces were applied to minimize blood contamination and clot formation as well as facile integration of hydrophilic patterns. This led to droplet compartmentalization and was utilized for detection of Interleukin 6 in a rapid dip-based assay. Furthermore, in a review article the need for anti-viral or virus repellent surfaces and future perspectives were discussed as the global COVID-19 pandemic surged and attracted interest toward innovative technologies for suppressing the spread of pathogens. To address the pressing issue of non-specific adhesion in diagnostics devices, an omniphobic liquid infused electrochemical biosensor was developed. This was achieved by electroplating gold nanostructures on fluorosilanized gold electrodes. These electrodes demonstrated rapid and specific detection of Escherichia coli within an hour in complex biological liquids (blood, urine, etc.) without dilutions or amplification steps from clinical patient samples which are major bottle necks when rapid detection systems are sought for at the point of care. / Thesis / Doctor of Philosophy (PhD) / Repellent surfaces have a variety of applications in healthcare, for coating medical devices (e.g. indwelling implants, stethoscopes, and other external devices.), coating hospital surfaces for blood and pathogen repellency, and for developing anti-fouling diagnostic devices. Furthermore, they can be applied in the food sector for limiting contaminations, and in public areas on high-touch surfaces to eliminate the spread of infection. Therefore, there is a need for repellent surface which can be easily applied to surfaces with various form factors while having an easy fabrication method. Featuring hierarchical structures on a heat-shrinkable material, a repellent wrap was designed to be integrated on existing surfaces and repel pathogens and suppress the spread of infection as an intermediate surface. Similar concept was used for designing blood repellent surfaces which were patterned with hydrophilic regions for a rapid dip-based biosensing platform. Finally, surface textures on conductive materials with liquid infused repellent coatings were investigated for electrochemical biosensing in complex biological liquids.

The research presented in this dissertation supports the overall goal of creating piezoelectric measurement technology for the analysis and characterization of soft materials that serve as feedstocks (inputs) and products (outputs) of emerging biomanufacturing processes, including cell and additive biomanufacturing processes. The first objective was to define measurement challenges associated with real-time monitoring of material compositional profiles using biosensors in practical biomanufacturing and bioprocessing formats, as insight into a material's composition (i.e., concentration of a given biologic within a material or product) provides molecular-scale insight into processes and product quality. The second objective was to design, fabricate, and characterize continuous flow cell separation technology based on 3D printed self-exciting and -sensing millimeter-scale piezoelectric transducers and microfluidic networks for separation and characterization of expanded therapeutic cells. The third objective was to establish a sensor-based characterization approach for viscoelastic properties of hydrogels and gelation processes using high-order modes of piezoelectric-excited millimeter cantilever (PEMC) sensors and understand the influence of cantilever mode number on critical sensor characteristics, including sensitivity, dynamic range, and limit of detection. The first objective was addressed through a comprehensive review of recent progress in electrochemical and hybrid biosensors, which included discussions of measurement formats, sensor performance, and measurement challenges associated with use in practical bioprocessing environments. This critical review revealed that cost, disposability, form factor, complex measurement matrices, multiplexing, and sensor regeneration/reusability are among the most pressing challenges that require solutions through advancement of sensor design and manufacturing approaches before biosensors can facilitate high-confidence long-term continuous bioprocess monitoring. The second objective was addressed by creating a microextrusion-based additive manufacturing approach for fabrication of piezoelectric-based MEMS devices that enabled integration of 3D configurations of piezoelectric transducers and microfluidic networks in a one-pot manufacturing process. The devices contained orthogonal out-of-plane piezoelectric sensors and actuators and generated tunable bulk acoustic waves (BAWs) capable of size-selective manipulation, trapping, and separation of suspended particles in droplets and microchannels. This work suggests that additive manufacturing potentially provides new opportunities for the fabrication of sensor-integrated microfluidic platforms for cell culture analysis. The third objective was addressed through resonant frequency tracking of low- and high-order modes in dynamic-mode cantilevers to enable the real-time characterization of hydrogel viscoelastic properties and continuous monitoring of sol-gel phase transitions over a wide dynamic range using practically relevant hydrogel systems used commonly in additive biomanufacturing. This work suggests that high-order modes of PEMC sensors facilitate characterization of hydrogel viscoelastic properties and gelation processes with improved dynamic range and limit of detection that can complement the performance of low-order modes. Through this research, new approaches for sensor-based characterization of soft material composition and mechanical properties using millimeter-scale piezoelectric devices are presented as solutions for current challenges in biomanufacturing and biosensing to advance capability in real-time sensing of quality attributes among biomanufactured products. / Doctor of Philosophy / The research presented in this dissertation supports the overall goal of creating sensor-based measurement technology for quality assessment of soft materials within practical online biosensing and biomanufacturing processing formats. This technology seeks to enable monitoring and control of product quality in real-time. Soft biomaterials used in these processes, including cells and hydrogels, can be characterized by quality signatures such as concentration of analytes and physical and mechanical properties. Separation and fluid handling technologies aid real-time characterization when integrated with the processing system. By improving sensor-based measurement capability of soft materials, sensing platforms can provide online quality assurance and control, thereby increasing the product quality and process efficiency – or yield– at reduced cost. The first objective was to define measurement challenges and limitations associated with detection of biologics in practical biomanufacturing and bioprocessing formats (with focus on pathogen detection, as the detection of adventitious agents and pathogens remains a critical aspect of bioprocess monitoring). This was addressed through a comprehensive review of recent progress in the field of electrochemical and hybrid biosensors. The second objective was to design and fabricate sensor-integrated microfluidic technology for cell separation applications using a combination of multi-material 3D printing and pick-and-place techniques. The third objective was to improve measurement capability of piezoelectric sensors for characterization of viscoelastic properties of hydrogel formulations commonly used in additive biomanufacturing processes and tissue engineering. Through this research, new approaches for sensor-based characterization of soft materials using millimeter-scale piezoelectric devices are presented as solutions for current challenges in biomanufacturing and biosensing platforms in order to advance quality assessment capability.

Le développement de méthodes rapides, précises et ultra-sensibles pour la détection d'analytes cibles en solution est crucial pour la recherche et les applications potentielles en médecine ou biologie moléculaire. Une approche très prometteuse consiste à développer des nano-capteurs à partir de nano-objets métalliques (NOM) qui présentent une résonance d'extinction dans leur réponse optique. Cette résonance nommée résonance de plasmon de surface localisée (RPSL) peut être ajustée spectralement en jouant sur la nature, la morphologie et l'environnement du NOM. Mesurer des modifications sur la RPSL de nano-objets individuels en présence d'analytes cibles doit permettre de s'affranchir des effets de moyennes dans les mesures d'ensemble. De plus, cela ouvre la voie vers le développement d'échantillons micrométriques pour des tests multicibles sans étiquette (« label-free »).Dans ce travail on a développé un nouveau dispositif expérimental basé sur la technique de spectroscopie à modulation spatiale (SMS) permettant de sonder la réponse optique de NOM individuels en milieu liquide. En parallèle des méthodes de synthèse ont été mises au point pour obtenir des échantillons sondes stables permettant des mesures sur NOM unique, en particulier sur des bipyramides d'or qui présentent de nombreuses qualités intrinsèques faisant d'elles de bonnes candidates pour le « bio-sensing ».Des mesures ont été réalisées dans des environnements d'indice variable et les changements détectés sont en bon accord avec les simulations théoriques. De plus, de nombreuses études ont été réalisées pour comprendre l'influence des nombreux paramètres agissant sur la réponse optique des systèmes étudiés / Advances in the development of rapid, accurate and highly sensitive methods for detecting target analytes in solution will provide crucial tools for research and applications in medicine and molecular biology. One of the currently most promising approaches is the development of nanosensors based on the localized surface plasmon resonance (LSPR) of noble metal nano-objects (MNOs), which is an optical response that depends on their size, shape, composition and local environment. The ability to measure the modification of the reponse of a single MNO in the presence of a target analyte would allow each object to act as an independent probe with increased sensitivity as the signal would be isolated from the averaging effects of ensemble measurements. Furthermore it would allow the development of micrometric, functionalized multiprobe samples for multitarget label-free assays.In this work, a novel experimental setup based on the spatial modulation spectroscopy (SMS) technique has been developed to measure the optical response of individual nano-objects in a liquid environment. In parallel, a new technique has also been developed to elaborate stable probes for measurements with the new setup, with a focus on gold bipyramids due to numerous qualities that make them excellent candidates for biosensing probes. The setup has been used to measure the response of individual objects in environments of different real refractive indices and the detected changes have been shown to be in good agreement with theoretical calculations. Numerical studies have also been performed to investigate the influence on the optical response of numerous factors encountered in the studied systems

La rhéologie et la photonique de divers systèmes biologiques complexes allant des protéines jusqu'aux bactéries et cellules ont été étudiées dans cette thèse. Ces travaux se basent sur deux grands thèmes, où le premier traite la modification des surfaces solides avec des molécules biologiques tandis que le second se concentre sur l'étude des effets des différentes drogues sur des cellules malignes, et non malignes par des techniques microscopiques complémentaires. Dans ce travail, des matrices orientées de films de polyélectrolytes/membrane pourpre ont été produites et étudiées en fonction de différentes conditions physico-chimiques. Des peptides spécifiques présentant de propriétés de reconnaissance de surface pour le ZnSe et le Si ont été isolées par la technologie de Phage Display. Le peptide de Si a été utilisé dans la détection des molécules avec une microcavité de silicium poreux, et ceci a montré un meilleur seuil de détection comparé à celui des autres méthodes classiques de fonctionnalisation. Le peptide spécifique de ZnSe a été utilisé afin de démontrer son utilité pour la préservation de l'activité et structure secondaire native des biomolécules adsorbées. Concernant les cellules, une différence de réponse, entre deux types de cellules épithéliales mammaires malignes MCF-7 et non-malignes HMEC184A1, sous traitement avec la curcumine, a été démontrée sur les cellules vivantes et fixées. Après, une évaluation des forces d'interaction entre un agent clinique anticancéreux cetuximab (CET) et EGFR (Epidermal Growth Factor Receptor) sur la surface des cellules de carcinome épithéliales A431 a été réalisé via la microscopie à force atomique en mode force. Une différence sur l'élasticité des cellules et sur les forces de liaison EGFR-CET a été notée quand le CET a été combiné avec d'autres drogues thérapeutiques. Les résultats de nos études d'imagerie fonctionnelle pourraient ouvrir de nouvelles voies dans la recherche de traitements contre le cancer. / The rheology and photonics of various complex biological systems ranging from proteins to bacteria and cells have been studied in this thesis. The work is organized around two major themes where the first one deals with surface modifications for adsorption of biological molecules while the second one focuses on comparative studies of non-malignant and cancerous cells under the effect of various drugs, using complementary microscopic techniques. In this work, oriented polyelectrolyte/purple membrane matrices have been produced and studied under different physico-chemical conditions. Peptides with surface recognition properties for the ZnSe and Si semiconductors have been isolated by Phage Display technology. The Si specific peptide has been used in detection of molecules with a porous silicon microcavity, providing a considerably enhanced detection resolution compared to traditional functionalization methods. The specific peptide of ZnSe has been used to demonstrate its utility in preservation of activity and native secondary structure of biomolecules in their adsorbed form. In the second part of my work concerning the cells, a different response (in morphology and elasticity) under treatment with curcumin, for two types of malignant MCF-7 and non-malignant HMEC184A1 mammary epithelial cells was demonstrated on living and fixed cells. Then, an evaluation of binding interactions between a clinical anticancer agent Cetuximab (CET) and the Epidermal Growth Factor Receptor (EGFR) on the surface of epithelial carcinoma A431 cells was performed via force mode atomic force microscopy. A difference was noted on the elasticity of cells and also on the EGFR-CET binding forces when CET was combined with other therapeutic drugs. The results of our functional imaging studies might open new avenues in the research for treatments against cancer.

Ce travail de thèse s’inscrit dans un projet plus large qui vise à développer avec le laboratoire Ampère et l’Etablissement Français du Sang un microsystème capable de réaliser un phénotypage plaquettaire pour le diagnostic de la thrombopénie néonatale. Ce microsystème doit permettre d’isoler les plaquettes du sang total et de détecter les antigènes plaquettaires présents à leur surface. L’isolation des plaquettes se fera grâce à un module de magnétophorèse et un module de diélectrophorèse. La détection sera électrochimique. Le cœur de ce travail de thèse a donc consisté à développer des nanoparticules magnétiques pour le module de magnétophorèse. Ces nanoparticules doivent permettre la capture spécifique des plaquettes et servir de marqueur pour la détection électrochimique. Pour ce faire, des nanoparticules magnétiques ont donc été doublement fonctionnalisées en une seule étape avec un anticorps anti-CD32 dirigé contre l’antigène CD32 présent à la surface des plaquettes et avec une molécule électroactive. Après optimisation des différents paramètres de greffage, les propriétés électrochimiques de ces particules ont été caractérisées. Leurs propriétés de bioreconnaissance ont été testées sur l’antigène purifié puis sur plaquettes entières. Enfin la faisabilité de la manipulation des structures nanoparticules/plaquettes par magnétophorèse avec des micro-aimants a été démontrée. / Fetal/neonatal alloimmune thrombocytopenia (F/NAIT) represents a great threat to new-borns or fetus. It occurs when a woman becomes alloimmunized against fetal platelet antigens. With the aim to improve fetal and neonatal survival, in collaboration with Ampere Laboratory and Etablissement Français du Sang, we plan at developing a Point-of-Care (POC) platform for platelet phenotyping. The final POC microsystem will be able to perform magnetophoresis and dielectrophoresis for platelets isolation from whole blood, and their selective electrochemical detection allowing for their phenotyping. The development of nanoparticles (NPs) with magnetic, electrochemical and bio-selection properties is a key issue. Herein, we have focused on the elaboration of magnetic NPs bearing 1) anti-CD32 antibody for specific interaction with CD32 antigen, which is present at the surface of platelets and 2) ferrocene carboxylic acid, an electroactive molecule for detection. To achieve this, the coupling reactions of this electroactive molecule and this antibody were optimized and a one-pot reaction for double functionalization was developed. The bioactivity of the immobilized antibody was tested at the molecular and cellular level. The dual-functionalized NPs voltammetric signals were also investigated. Finally the feasibility of platelets capture and actuation by magnetophoresis with micro-magnet array were demonstrated.

Arsenic contamination of groundwater remains a serious health concern in many areas of the world. Developing countries such as Bangladesh and Nepal are particularly affected because access to high quality water infrastructure is low. Since the 1970s, most water in these countries is sourced from shallow tube wells installed to reduce the spread of diseases associated with poor water hygiene. In this goal they were successful, however by the mid 1990s it became apparent that many of these wells were contaminated by arsenic and that these countries’ rural poor were being slowly poisoned. No simple, cheap, and reliable test for arsenic exists, and efforts to mitigate arsenic contamination have been severely limited by this over the past two decades. Government backed well-testing efforts using commercially available field kits have many issues with reliability, safety, rigour, and transparency, and have lost their urgency over the past decade, while the expensive field test kits remain out of the reach of most ordinary people in these areas. Synthetic Biology offers the technology to develop a new class of biosensor by exploiting bacteria’s natural ability to sense and respond to levels of arsenic considerably lower than commercially available kits which are based on analytical chemistry. In order to reach this goal, we must first develop our understanding of the natural response to arsenic in our chosen host, B. subtilis. Although we have a reasonably good qualitative understanding of the operon responsible for arsenic sensing, very little quantitative analysis has been carried out, and a robust system for ratiometric characterisation has not been established in the bacteria. In this work, a robust platform for rapid ratiometric characterisation is established in B. subtilis. A rigorous mathematical model of the ars operon is developed and analysed before being verified experimentally. This new knowledge is then used to explore synthetic permutations to the natural system aimed at improving the sensor properties of the system. Finally, a biological architecture for an easily tunable biosensor with good characteristics is recommended.