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No. of bitstreams: 2 GABRIEL_Gabriele_2017.pdf: 4796378 bytes, checksum: b47c6fae3bd085c69ed8a81c74a9be4d (MD5) GABRIEL_Gabriele_carta.pdf: 557241 bytes, checksum: fbed02fe69c0cab6190201e18b304f40 (MD5) Previous issue date: 2017-12-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Bioluminescence, the emission of visible light by living organisms is widely used in biosensors. Firefly luciferases and genes are among the most used reporter gene in bioluminescent biosensors. Firefly luciferases are pH-sensitive, exhibiting a red shifted bioluminescence spectra on the presence of metals, high temperatures and acidic pH, being this last property considered unusual for the most analytical applications. Nowadays most luminescent biosensors to metals and pH are fluorescent, and few of them are ratiometric based on spectral changes. Bioluminescent biosensors despite been less common, have same advantages as low background and does not need UV light irradiation, bring no photodamage to the cell. The aim of this project was: (I) study the applicability of the use of Macrolampis sp2 firefly luciferase and other pH-sensitive luciferases as spectral intracellular ratiometric pH biosensor; (II) apply these pH ratiometric biosensors in mammalian cells to investigate its physiology in real time and (III) developing, by engineering the pH sensor region of Macrolampis sp2 firefly luciferase, a ratiometric biosensor specific to metals. We obtained a relation between the pH and the ratio of the bioluminescence at green and red region of the spectra, allowing estimate ratiometrically the intracellular pH of bacteria and mammalian cells. Besides that, we confirmed its use to cellular image of pH and observed that occurs an alkalinization of the cytosol and nucleus during cell division and apoptosis. We demonstrate the applicability of firefly luciferases, in special the luciferase of Macrolampis sp2, as a ratiometric biosensor to metals and intracellular pH. The existence of a linear relationship between the concentrations of metals like cadmium, mercury and zinc, and the ratio of the bioluminescence at green and red region of the spectra, allowed also estimate ratiometrically, for the first time, concentrations of metals less than 100 µM, enabling the use of firefly luciferases as bioavailability indicator to toxic and potential toxic metals. / Bioluminescência, a emissão de luz fria e visível por organismos vivos é amplamente utilizada em biossensores. Luciferases de vagalumes e seus genes estão entre os genes repórteres mais utilizados em biossensores bioluminescentes. Luciferases de vagalumes são sensíveis ao pH, apresentando um deslocamento do espectro de bioluminescência para o vermelho na presença de metais, em temperaturas elevadas e pH ácido, sendo esta última propriedade considerada sem utilidade para a maioria das aplicações analíticas. Atualmente a maioria dos biossensores luminescentes para metais e pH são fluorescentes, poucos deles são raciométricos baseados nas mudanças espectrais. Biossensores bioluminescentes apesar de serem menos comuns, possuem algumas vantagens como baixo background e não necessitam de irradiação com luz UV, não causando fotodanos às células. Os objetivos desse projeto foram: (I) investigar a aplicabilidade de uso da luciferase do vagalume Macrolampis sp2 e outras luciferases pH-sensitivas como biossensor espectral raciométrico intracelular de pH; (II) aplicar esses biossensores raciométricos de pH em células de mamíferos para investigar sua fisiologia em tempo real e (III) desenvolver, por engenharia da região sensora de pH da luciferase de Macrolampis sp2, um biossensor raciométrico específico para metais. Obtivemos uma relação entre o pH e a razão da bioluminescência nas regiões do verde e do vermelho do espectro, permitindo estimar raciometricamente o pH intracelular de bactérias e células de mamíferos. Além disso, confirmamos seu uso para imagem celular de pH, e observamos que ocorre uma alcalinização do citosol e núcleo durante os processos de divisão celular e apoptose. Demonstramos a aplicabilidade das luciferases de vagalumes, em especial a luciferase de Macrolampis sp2, como biossensor raciométrico para metais e pH intracelular. A existência de uma relação linear entre a concentração de metais como cádmio, mercúrio e zinco, e a razão da bioluminescência nas regiões do verde e do vermelho do espectro, permitiu também estimar raciometricamente pela primeira vez concentrações de metais menores que 100 µM, possibilitando o uso de luciferase de vagalumes como indicador de biodisponibilidade de metais tóxicos e potencialmente tóxicos. / FAPESP: 2014/04477-9 / FAPESP: 2015/22603-4 / FAPESP: 2016/15946-5

2D molybdenum disulfide (MoS₂) gives a new inspiration for the field of nanoelectronics, photovoltaics, and sensorics. However, the most common processing technology, e.g., liquid‐phase based scalable exfoliation used for device fabrication, leads to the number of shortcomings that impede their large area production and integration. Major challenges are associated with the small size and low concentration of MoS₂ flakes, as well as insufficient control over their physical properties, e.g., internal heterogeneity of the metallic and semiconducting phases. Here it is demonstrated that large semiconducting MoS₂ sheets (with dimensions up to 50 µm) can be obtained by a facile cathodic exfoliation approach in nonaqueous electrolyte. The synthetic process avoids surface oxidation thus preserving the MoS₂ sheets with intact crystalline structure. It is further demonstrated at the proof‐of‐concept level, a solution‐processed large area (60 × 60 µm) flexible Ebola biosensor, based on a MoS₂ thin film (6 µm thickness) fabricated via restacking of the multiple flakes on the polyimide substrate. The experimental results reveal a low detection limit (in femtomolar–picomolar range) of the fabricated sensor devices. The presented exfoliation method opens up new opportunities for fabrication of large arrays of multifunctional biomedical devices based on novel 2D materials.

We report on the spatial and temporal signaling properties of a yeast pheromone-based cell communication and amplifier system. It utilizes the Saccharomyces cerevisiae mating response pathway and relies on diffusion of the pheromone α–factor as key signaling molecule between two cell types. One cell type represents the α–factor secreting sensor part and the other the reporter part emitting fluorescence upon activation. Although multi-cellular signaling systems promise higher specificity and modularity, the complex interaction of the cells makes prediction of sensor performance difficult. To test the maximum distance and response time between sensor and reporter cells, the two cell types were spatially separated in defined compartments of agarose hydrogel (5 ´ 5 mm) and reconnected by diffusion of the yeast pheromone. Different ratios of sensor to reporter cells were tested to evaluate the minimum amount of sensor cells required for signal transduction. Even the smallest ratio, one α–factor-secreting cell to twenty reporter cells, generated a distinct fluorescence signal. When using a 1:1 ratio, the secreted pheromone induced fluorescence in a distance of up to four millimeters after six hours. We conclude from both our experimental results and a mathematical diffusion model that in our approach: (1) the maximum dimension of separated compartments should not exceed five millimeters in gradient direction; and (2) the time-limiting step is not diffusion of the signaling molecule but production of the reporter protein.

This work was performed in the framework of two application-oriented research projects that focus on the generation and evaluation of fluorescent Saccharomyces (S.) cerevisiae-based sensor and reporter cells for white biotechnology as well as the extension of the conventional single-cell/single-construct principle of ordinary yeast biosensor approaches. Numerous products are currently generated by biotechnological processes which require continuous and precise process control and monitoring. These demands are only partially met by physical or physiochemical sensors since they measure parameters off-line or use surrogate parameters that consequently provide only indirect information about the actual process performance. Biosensors, in particular whole cell-based biosensors, have the unique potential to near-line and long-term monitor parameters such as nutrient availability during fermentation processes. Moreover, they allow for the assessment of an analyte’s biological relevance. Prototype yeast sensor and reporter strains derived from common laboratory strains were transformed with multicopy expression plasmids that mediate constitutive or inducible expression of a fluorescence reporter gene. Performance of these cells was examined by various qualitative and quantitative detection methods – representative of putative transducer technologies. Analyses were performed on the population level by microplate reader-based fluorometry and Western blot as well as on the single-cell level by fluorescence microscopy and flow cytometry. ‘Signature’ promoters that are activated or repressed during particular nutrient-limited growth conditions were selected in order to generate yeast nutrient sensor strains for monitoring the biological availability of nitrogen, phosphorus or sulphur. For each category, at least one promoter mediating at least threefold changed green fluorescence levels between sensor cells in non-limited and nutrient-limited conditions was identified. Sensor strains were evaluated in detail regarding sensitivity, analyte selectivity and the ability to restore basic fluorescence after shift from nutrient-limited to non-limited conditions (regeneration). The applicability for bioprocess monitoring purposes was tested by growth of yeast nutrient sensor cells in microalgae media and supernatants. Despite successful proof of principle, numerous challenges still need to be solved to realise prospective implementation in this field of white biotechnology. The major drawback of plasmid-borne detection constructs is a high fluorescence variance between individual cells. By generation of a nitrogen sensor strain with a genome-integrated detection construct, uniform expression on the single-cell level and simultaneous maintenance of basic properties (ability of fluorescence induction/regeneration and lack of cross-reactivity) was achieved. However, due to the singular detection construct per cell, significantly weaker overall fluorescence was observed. The traditional single-cell/single-construct approach was expanded upon in two ways. Firstly, a practical dual-colour sensor strain was created by simultaneous, constitutive expression of a red fluorescence reporter gene in green fluorescent nitrogen sensor cells. Secondly, an innovative cellular communication and signal amplification system inspired by the natural S. cerevisiae pheromone system and mating response was established successfully. It features the yeast pheromone alpha-factor as a trigger and alpha-factor-responsive reporter cells which express a fluorescence reporter gene from the pheromone-inducible FIG1 promoter as an output signal. The system was functional both with synthetic and cell-secreted alpha-factor, provided that recombinant cells were deleted for the alpha-factor protease Bar1p. Integration of amplifier cells which secrete alpha-factor in response to stimulation with the pheromone itself could increase the system\'s sensitivity further. Signal amplification was demonstrated for phosphorus sensor cells as a proof of concept. Therefore, the alpha-factor-based cellular communication and signal amplification system might be useful in applications that suffer from poor signal yield. Due to its modular design, the system could be applied in basically any cell-based biosensor or sensor-actor system. Immobilisation of the generated sensor and reporter cells in transparent natural polymers can be beneficial considering biosensor fabrication. Functionality of sensor and reporter cells in calcium-alginate beads or nano-printed arrays was successfully demonstrated. For the latter setup, fluorescence scanning and software-assisted fluorescence quantification was applied as a new detection method. In an experiment using an agarose-based two-compartment setup proposed by Jahn, 2011, properties of the alpha-factor-based cellular communication and signal amplification system after immobilisation were tested. These studies provide an initial experimental basis for an appropriate geometry of miniaturised immobilisation matrices with fluorescent yeast sensor and reporter cells in prospective biosensor designs.

It is of paramount importance to our society that the environment, life style, science and amusement flourish together in a balanced way. Some trends in this direction are the increased utilization of renewable energy, like solar photovoltaics; better health care products, for example advanced biosensors; high definition TV or high resolution cameras; and novel scientific tools for better understanding of scientific observations. Advancement of micro and nanotechnologies has directly and positively impacted our stance in these application domains; one example is that of vertical periodic or aperiodic nano or micro pillar assemblies which have attracted significant research and industrial interest in recent years. In particular, Si pillars are very attractive due to the versatility of silicon. There are many potential applications of Si nanopillar/nanowire assemblies ranging from light emission, solar cells, antireflection, sensing and nonlinear optical effects. Compared to bulk, Si pillars or their assemblies have several unique properties, such as high surface to volume ratios, light localization, efficient light guiding, better light absorption, selective band of light propagation etc.      The focus of the thesis is on the fabrication of Si pillar assemblies and hierarchical ZnO nanowires on Si micro structures in top-down and bottom-up approaches and their optical properties and different applications. Here, we have investigated periodic and aperiodic Si nano and micro structure assemblies and their properties, such as light propagation, localization, and selective guiding and light-matter interaction. These properties are exploited in a few important optoelectronic/photonic applications, such as optical biosensors, broad-band anti-reflection, radial-junction solar cells, second harmonic generation and color filters.         We achieved a low average reflectivity of ~ 2.5 % with the periodic Si micropyramid-ZnO NWs hierarchical arrays. Tenfold enhancement in Raman intensity is also observed in these structures compared to planar Si. These Si microstructure-ZnO NW hierarchical structures can enhance the performance and versatility of photovoltaic devices and optical sensors. A convenient top-down fabrication of radial junction nanopillar solar cell using spin-on doping and rapid thermal annealing process is presented. Broad band suppressed reflection, on average 5%, in 300- 850 nm wavelength range and an un-optimized cell efficiency of 6.2 % are achieved. Our method can lead to a simple and low cost process for high efficiency radial junction nanopillar solar cell fabrication.            Silicon dioxide (SiO2) coated silicon nanopillar (NP) arrays are demonstrated for surface sensitive optical biosensing. Bovine serum albumin (BSA)/anti-BSA model system is used for biosensing trials by photo-spectrometry in reflection mode. Best sensitivity in terms of limit of detection of 5.2 ng/ml is determined for our nanopillar biosensor. These results are promising for surface sensitive biosensors and the technology allows integration in the CMOS platform.         Si pillar arrays used for surface second harmonic generation (SHG) experiments are shown to have a strong dependence of the SHG intensity on the pillar geometry. The surface SHG can be suitable for nonlinear silicon photonics, surface/interface studies and optical sensing.         Aperiodic Si nanopillar assemblies in PDMS matrix are demonstrated for efficient color filtering in transmission mode. These assemblies are designed using the ‘‘molecular dynamics-collision between hard sphere’’ algorithm. The designed structure is modeled in a 3D finite difference time domain (FDTD) simulation tool for optimization of color filtering properties. Transverse localization effect of light in our nanopillar color filter structures is investigated theoretically and the results are very promising to achieve image sensors with high pixel densities (~1 µm) and low crosstalk. The developed color filter is applicable as a stand-alone filter for visible color in its present form and can be adapted for displays, imaging, smart windows and aesthetic applications. / <p>QC 20160407</p>

Characterizing nanoparticles to be able to understand how they functions in the body is important for development of drugs. Furthermore with increasing number of nanoparticle product the nanotoxicity of nanoparticles is important to understand. This report is a part of the EU-project Nanoclassifier which purpose is to “develop a cost effective, high throughput screening platform for characterization of the bionanointerface and its cell-binding partners”. Single-cycle kinetic was used to determine the number of binding epitopes on polystyrene nanoparticle with transferrin corona. The number of available epitopes describes how active the Nanoparticle will be in the body. For this purpose Single-cycle kinetic methodology was successfully used on nanoparticles. Single-cycle kinetic methodology has great potential to become the standard method for high throughput nanoparticle epitope characterization.

Surface-plasmon-resonance (SPR) sensors are widely used in biological, chemical, medical, and environmental sensing. This dissertation describes the design and development of dual-mode, self-referencing SPR sensors supporting two surface-plasmon modes (long- and short-range) which can differentiate surface binding interactions from bulk index changes at a single sensing location. Dual-mode SPR sensors have been optimized for surface limit of detection (LOD). In a wavelength interrogated optical setup, both surface plasmons are simultaneously excited at the same location and incident angle but at different wavelengths. To improve the sensor performance, a new approach to dual-mode SPR sensing is presented that offers improved differentiation between surface and bulk effects. By using an angular interrogation, both surface plasmons are simultaneously excited at the same location and wavelength but at different angles. Angular interrogation offers at least a factor of 3.6 improvement in surface and bulk cross-sensitivity compared to wavelength-interrogated dual-mode SPR sensors. Multi-mode SPR sensors supporting at least three surface-plasmon modes can differentiate a target surface effect from interfering surface effects and bulk index changes. This dissertation describes a tri-mode SPR sensor which supports three surface plasmon resonance modes at one single sensing position, where each mode is excited at a different wavelength. The tri-mode SPR sensor can successfully differentiate specific binding from the non-specific binding and bulk index changes.

The use of microfluidic tools to develop two novel approaches to surface-based oligonucleotide hybridization assays has been explored. In one of these approaches, immobilized oligonucleotide probes on a glass surface of a microfluidic channel were able to quantitatively hybridize with oligonucleotide targets that were electrokinetically injected into the channel. Quantitative oligonucleotide analysis was achieved in seconds, with nM detection limits and a dynamic range of 3 orders of magnitude. Hybridization was detected by the use of fluorescently labeled target. The fluorescence intensity profile evolved as a gradient that could be related to concentration, and was a function of many factors including hybridization reaction rate, convective delivery speed, target concentration and target diffusion coefficient. It was possible to acquire kinetic information from the static fluorescence intensity profile to distinguish target concentration, and the length and base-pair mismatches of target sequences. Numerical simulations were conducted for the system, and fit well with the experimental data. In a second approach, a solid-phase nucleic acid assay was developed using immobilized Quantum Dot (QD) bioprobes. Hybridization was used to immobilize QDs that had been coated with oligonucleotides having two different sequences. The hybridization of one oligonucleotide sequence conjugated to a QD (a linker sequence) with a complementary sequence that was covalently attached to a glass substrate of a microfluidic channel was shown to be an immobilization strategy that offered flexibility in assay design, with intrinsic potential for quantitative replacement of the sensing chemistry by control of stringency. A second oligonucleotide sequence conjugated to the immobilized QDs provided for the selective detection of target nucleic acids. The microfluidic environment offered the ability to manipulate flow conditions for control of stringency and increasing the speed of analytical signal by introduction of convective delivery of target sequences to the immobilized QDs. This work introduces a stable and adaptable immobilization strategy that facilitates solid-phase QD-bioprobe assays in microfluidic platforms.

Many biological processes, pathogens, and pharmaceuticals act upon, cellular membranes. Accordingly, cell membrane mimics are attractive targets for biosensing, with research, pathology, and pharmacology applications. Lipid bilayers represent a versatile sensor functionalization platform providing antifouling properties, and many receptor integration options, uniquely including transmembrane proteins. Bilayer-coated sensors enable the kinetic characterization of membrane/analyte interactions. Addressed theoretically and experimentally is the self-assembly of model membranes on plasmonic sensors. Two categories of plasmonic sensors are studied in two parts. Part I aims to deposit raft-forming bilayers on planar nanoaperture arrays suitable for multiplexing and device integration. By vesicle fusion, planar bilayers are self-assembled on thiol-acid modified flame-annealed gold without the need for specific lipid head-group requirements. Identification of coexisting lipid phases is accomplished by AFM imaging and force spectroscopy mapping. These methods are successfully extended to metallic, plasmon-active nanohole arrays, nanoslit arrays and annular aperture arrays, with coexisting phases observed among the holes. Vis-NIR transmission spectra of the arrays are measured before and after deposition, indicating bilayer detection. Finally, the extraction of membrane proteins from cell cultures and incorporation into model supported bilayers is demonstrated. These natural membrane proteins potentially act as lipid-bound surface receptors. Part II aims to encapsulate in model lipid bilayers, metallic nanoparticles, which are used as probes in surface enhanced Raman spectroscopy. Three strategies of encapsulating particles, and incorporating Raman-active dyes are demonstrated, each using a different dye: malachite green, rhodamine-PE, and Tryptophan. Dye incorporation is verified by SERS and the bilayer is visualized and measured by TEM, with support from DLS and UV-Vis spectroscopy. In both parts, lipid-coated sensors are successfully fabricated and characterized. These results represent important and novel solutions to the functionalization of plasmonic surfaces with biologically relevant cell membrane mimics.

This thesis is focused on enzymatic studies of the secreted aspartic proteases (Saps) from Candida albicans as a tool for discovery of anti-candida drugs. C. albicans causes infections in a number of different locations, which differ widely in the protein substrates available and pH. Since C. albicans needs Saps during virulent growth, these enzymes are good targets for drug development. In order to investigate the catalytic characteristics of Saps and their inhibitor affinities, substrate-based kinetic assays were developed. Due to the low sensitivity of these assays, especially at the sub-optimal pH required to mimic the different locations of infections, these assays were not satisfactory. Therefore, a biosensor assay was developed whereby, it was possible to study interaction between Saps and inhibitors without the need to optimise catalytic efficacy. Furthermore, the biosensor assay allowed determination of affinity, as well as the individual association and dissociation rates for inhibitor interactions. Knowledge about substrate specificity, Sap subsite adaptivity, and the pH dependencies of catalytic efficacy has been accumulated. Also, screening of transition-state analogue inhibitors designed for HIV-1 protease has revealed inhibitors with affinity for Saps. Furthermore, the kinetics and pH dependencies of their interaction with Saps have been investigated. One of these inhibitors, BEA-440, displayed a complex interaction with Saps, indicating a conformational change upon binding and a very slow dissociation rate. A time dependent interaction was further supported by inhibition measurements. The structural information obtained affords possibilities for design of new more potent inhibitors that might ultimately become drugs against candidiasis. The strategy to combine substrate specificity studies with inhibitor screening has led to complementary results that generate a framework for further development of potent inhibitors.