1,089 research outputs found
Gold nanorods functionalized with DNA oligonucleotide probes for biosensing and plasmon-enhanced fluorescence detection
Gold nanorods display plasmon resonances that are very sensitive to the refraction index
close to the particle’s surface. The site-selective functionalization of Plasmon hot-spots with
bioreceptors is crucial to develop plasmonic sensors with improved response bycapturing the
target species at the most sensitive regions of the particle. Firstly, we used surface
immobilized biotin-functionalized gold nanorods for streptavidin sensing.The selective
functionalization of the nanorods’ tips was achieved with a CTAB bilayer and using a thiol
linker to attach the desired biotin functionality. The sensor performance was characterized by
measuring binding kinetic assays. In the recent years, Dengue virus DENV-2 has been
reported as the largest dengue epidemic type and early stage detection of this virus would
save the life of many patients. Thus, a plasmonic model biosensor was designed for the
detection of RNA sequences proposed as disease biomarkers for Dengue virus.For this
purpose, we have functionalized gold nanorods with thiolated DNA oligonucleotide probes
complementary to a RNA sequence of Dengue virus.As a signal amplification strategy, we
have used biotin-labeled oligonucleotide target sequences, in order to bind streptavidin or
anti-biotin antibody to increase the surface plasmon response.
Plasmon-enhanced fluorescence (PEF) microscopy provides fast, high-contrast, and lowbackground
detection of single molecules. The interaction between the localized surface
plasmon of gold nanorods and a fluorophore in their vicinity can induce the acceleration of
excitation and decay rates thus leading to substantial fluorescence enhancements. In the third
part of this Thesis, it was studied the interaction between gold nanorod antennas and a weakly
fluorescence dye, TMPyP porphyrin. This interaction was mediated by electrostatic attraction
between the tetracationic TMPyP and the DNA oligonucleotide coating on the nanorods’
surface.
Preliminary measurements of optical spectroscopy were carried out to characterize the
interaction in solution of TMPyP and single or double-stranded DNA oligonucleotides
complementary to a RNA sequence of Dengue virus.The apparent equilibrium constants for
the complex of TMPyP with single and double-stranded DNA were determined to be Ka=
3.9×107 M-1and 4.5×107 M-1respectively. The spectral changes show a strong specific
intercalation of TMPyP with ds-DNA and ss-DNA because of GC-rich sites in the selected
sequences. Next, the plasmon-enhanced fluorescence of TMPyP induced by gold nanorods
was investigated using confocal fluorescence lifetime microscopy to perform measurements
of nanoparticle emission intensity and spectrum, fluorescence correlation spectroscopy,
emission intensity time trace and fluorescence decay. The gold nanorods were immobilized
on glass and functionalized with a thiolated oligonucleotide coating, while TMPyP molecules
are diffusing in solution and stochastically interact with the rod’s surface. The emission
intensity traces measured on single particles show strong fluorescence bursts when TMPyP
molecules come into close proximity of the nanorod. We have calculated the emission
enhancement factors from a comparison with the non-enhanced emission of TMPyP in the
same experimental conditions and found surprisingly large enhancement factors of around
60000-fold for TMPyP’s emission.These values of enhancement are two orders of magnitude
larger than our calculated highest enhanced fluorescence expected for TMPyP molecule.Os nano-bastonetes de ouro são caracterizados por plasmões de superfície com frequências de
ressonância bastante sensíveis ao índice de refração na proximidade da sua superfície. A
funcionalização seletiva da superfície destas nanopartículas com bio-receptores é crucial para
o desenvolvimento de sensores plasmónicos com resposta melhorada, pois permite a captura
de analitos nas regiões mais sensíveis da nanopartícula. Em primeiro lugar foram preparadas
superfícies com nano-bastonetes de ouro que depois foram funcionalizados com recetores
biotina para ensaios modelo de deteção de estreptavidina. A funcionalização seletiva das
extremidades dos nano-bastonetes foi conseguida através da proteção das suas paredes
laterais com uma bicamada de tensioativo CTAB e usando uma biotina derivatizada com uma
função tiól. O desempenho do sensor foi caracterizado por medidas da cinética de associação
biotina-estreptavidina monitorizada por espectroscopia ótica de absorção. Em anos recentes, a
infeção pelo vírus do Dengue DENV-2 tem sido relatada como a maior epidemia por este tipo
de vírus, e a deteção precoce desta infeção poderia salvar a vida de muitos pacientes. Deste
modo, foi desenhado um sensor plasmónico modelo para a deteção de sequências de ARN
propostas como bio-marcadores para a infeção pelo vírus do Dengue. Para o efeito, foram
funcionalizados nano-bastonetes de ouro com cadeias de oligonucleotídos de ADN
complementares a uma sequência do ARN do vírus do Dengue. Como estratégia de
amplificação de sinal foram usadas cadeias de oligonucleotídos alvo marcadas com biotina,
de modo a ser possível num segundo passo ligar estreptavidina ou anticorpo anti-biotina com
o objetivo de aumentar a resposta do plasmão de superfície dos nano-bastonetes de ouro.
A fluorescência intensificada por efeito plasmónico permite a deteção rápida e com elevado
contraste de molécula única em microscopia de fluorescência. A interação entre os modos
localizados de plasmão de superfície de nano-bastonetes de ouro e moléculas fluorescentes na
sua proximidade pode induzir a aceleração das taxas de excitação, decaimento radiativo e
não-radiativo, e conduzir a uma intensificação de fluorescência.Na terceira parte desta
Dissertação, foram investigadas as interações entre nano-antenas de ouro e um cromóforo
pouco fluorescente, a porfirina TMPyP. Esta interação foi mediada pela atração eletrostática
entre a porfirina tetra-catiónica e o revestimento de ADN na superfície dos nano-bastonetes
de ouro.
Ensaios preliminares de espectroscopia ótica foram realizados para caracterizar a interação
em solução da TMPyP com sequências de ADN de cadeia simples ou duplacomplementares a
uma sequência do ARN do vírus do Dengue. A constante aparente de equilíbrio para o
complexo da TMPyP com as sequências de ADN de cadeia simples e dupla foram
determinadas como sendo Ka= 3.9×107 M-1and 4.5×107 M-1, respetivamente. As alterações
dos espectros de absorção e emissão mostram uma forte interação, provavelmente
intercalação, daTMPyPcom ods-DNA,etambém com o ss-DNA, devido ao elevado conteúdo
em pares GC nas sequências escolhidas. Em seguida, a fluorescência intensificada por efeito
plasmónico na TMPyP induzida por nano-bastonetes de ouro foi investigada por microscopia
confocal de tempos-de-vida, tendo sido realizadas medidas de intensidade e espectro de
emissão de nanopartículas, espectroscopia de correlação de fluorescência, traços temporais de
intensidade de emissão e de decaimento de fluorescência.Os nano-bastonetes de ouro foram
imobilizados em vidro e funcionalizados com um revestimento de oligonucleotídostiolados,
enquanto que as moléculas de TMPyP difundem-se em solução e podem interatuar
estocasticamente com a superfície da nanopartícula. Os traços de intensidade de emissão
medidos em nanopartículas individuais mostram picos de fluorescência intensos quando as
moléculas de TMPyP se aproximam do nano-bastonete de ouro em resultado do efeito de
nano-antena.Foram calculados os fatores de emissão intensificada por comparação com a
emissão não-intensificada da TMPyP nas mesmas condições experimentais e obtiveram-se
valores surpreendentemente elevados de cerca de 60000 vezes para a emissão intensificada da
TMPyP. Estes fatores de intensificação são duas ordens de grandeza mais elevados do que as
estimativas teóricas calculadas para a intensificação da emissão da TMPyP pelos nanobastonetes
de ouro
Provenance does matter: links between winter trophic segregation and the migratory origins of European robins
Amongst migratory species, it is common to find individuals from different populations or geographical origins sharing staging or wintering areas. Given their differing life histories, ecological theory would predict that the different groups of individuals should exhibit some level of niche segregation. This has rarely been investigated because of the difficulty in assigning migrating individuals to breeding areas. Here, we start by documenting a broad geographical gradient of hydrogen isotopes (δ2H) in robin Erithacus rubecula feathers across Europe. We then use δ2H, as well as wing-tip shape, as surrogates for broad migratory origin of birds wintering in Iberia, to investigate the ecological segregation of populations. Wintering robins of different sexes, ages and body sizes are known to segregate between habitats in Iberia. This has been attributed to the despotic exclusion of inferior competitors from the best patches by dominant individuals. We find no segregation between habitats in relation to δ2H in feathers, or to wing-tip shape, which suggests that no major asymmetries in competitive ability exist between migrant robins of different origins. Trophic level (inferred from nitrogen isotopes in blood) correlated both with δ2H in feathers and with wing-tip shape, showing that individuals from different geographic origins display a degree of ecological segregation in shared winter quarters. Isotopic mixing models indicate that wintering birds originating from more northerly populations consume more invertebrates. Our multi-scale study suggests that trophic-niche segregation may result from specializations (arising in the population-specific breeding areas) that are transported by the migrants into the shared wintering grounds
Decomposition of stochastic flows with automorphism of subbundles component
We show that given a -structure on a differentiable manifold , if
the group of automorphisms of is big enough, then there exists the
quotient of an stochastic flows by , in the sense that where , the remainder has
derivative which is vertical but transversal to the fibre of . This
geometrical context generalizes previous results where is a Riemannian
manifold and is decomposed with an isometric component, see Liao
\cite{Liao1} and Ruffino \cite{Ruffino}, which in our context corresponds to
the particular case of an SO(n)-structure on .Comment: To appear in Stochastics and Dynamics, 201
Shape acquisition of rotating objects based on Laser Line Scanning
The present work proposes a methodology for 3D shape acquisition of objects through rotation-based Laser Line Scanning. This
enables the acquisition of an object's 3D shape from multiple views,
which leads to a more complete and accurate model
Grid structure impact in sparse point representation of derivatives
In the Sparse Point Representation (SPR) method the principle is to retain the function data indicated by significant interpolatory wavelet coefficients, which are defined as interpolation errors by means of an interpolating subdivision scheme. Typically, a SPR grid is coarse in smooth regions, and refined close to irregularities. Furthermore, the computation of partial derivatives of a function from the information of its SPR content is performed in two steps. The first one is a refinement procedure to extend the SPR by the inclusion of new interpolated point values in a security zone. Then, for points in the refined grid, such derivatives are approximated by uniform finite differences, using a step size proportional to each point local scale. If required neighboring stencils are not present in the grid, the corresponding missing point values are approximated from coarser scales using the interpolating subdivision scheme. Using the cubic interpolation subdivision scheme, we demonstrate that such adaptive finite differences can be formulated in terms of a collocation scheme based on the wavelet expansion associated to the SPR. For this purpose, we prove some results concerning the local behavior of such wavelet reconstruction operators, which stand for SPR grids having appropriate structures. This statement implies that the adaptive finite difference scheme and the one using the step size of the finest level produce the same result at SPR grid points. Consequently, in addition to the refinement strategy, our analysis indicates that some care must be taken concerning the grid structure, in order to keep the truncation error under a certain accuracy limit. Illustrating results are presented for 2D Maxwell's equation numerical solutions
Live-cell FRET imaging reveals clustering of the prion protein at the cell surface induced by infectious prions
Prion diseases are associated to the conversion of the prion protein into a misfolded pathological isoform. The mechanism of propagation of protein misfolding by protein templating remains largely unknown. Neuroblastoma cells were transfected with constructs of the prion protein fused to both CFP-GPI-anchored and to YFP-GPI-anchored and directed to its cell membrane location. Live-cell FRET imaging between the prion protein fused to CFP or YFP was measured giving consistent values of 10 +/- 2%. This result was confirmed by fluorescence lifetime imaging microscopy and indicates intermolecular interactions between neighbor prion proteins. In particular, considering that a maximum FRET efficiency of 17 +/- 2% was determined from a positive control consisting of a fusion CFP-YFP-GPI-anchored. A stable cell clone expressing the two fusions containing the prion protein was also selected to minimize cell-to-cell variability. In both, stable and transiently transfected cells, the FRET efficiency consistently increased in the presence of infectious prions - from 4 +/- 1% to 7 +/- 1% in the stable clone and from 10 +/- 2% to 16 +/- 1% in transiently transfected cells. These results clearly reflect an increased clustering of the prion protein on the membrane in the presence of infectious prions, which was not observed in negative control using constructs without the prion protein and upon addition of non-infected brain. Our data corroborates the recent view that the primary site for prion conversion is the cell membrane. Since our fluorescent cell clone is not susceptible to propagate infectivity, we hypothesize that the initial event of prion infectivity might be the clustering of the GPI-anchored prion protein. (C) 2014 Elsevier B.V. All rights reserved.Fundacao para a Ciencia e Tecnologia (FCT), Portugal [PTDC/QUI-BIQ/119677/2010, PTDC/CTM-NAN/2700/2012]; FCT [PEst-OE/EQB/LA0023/2011, SFRH/BD/48664/2008, SFRH/BPD/64932/2009]info:eu-repo/semantics/publishedVersio
Biodiversity patterns of cavernicolous ground-beetles and their conservation status in the Azores, with the description of a new species: Trechus isabelae n. sp (Coleoptera : Carabidae : Trechinae)
Copyright © 2007 · Magnolia Press.Diversity patterns of cave and epigean Trechinae (Coleoptera, Carabidae) from the Azores (Portugal) are reported based on recently standardized sampling protocols in different habitats of this geologically young and isolated volcanic archipelago. A total of 10 species are studied, including Trechus isabelae n. sp., collected in a volcanic pit on São Jorge, one of the nine islands of the Azores. This new Trechus species represents the eighth species of Trechinae described from the underground environment of the Azores. An identification key for the Azorean species of Trechus is provided along with additional information per species on their distribution and conservation status in the archipelago. Possible reasons for the different degrees of adaptation to the conditions of the underground environment exhibited by Trechinae are also discussed
Inventory of tiger- and ground-beetles (Coleoptera Caraboidea: Cicindelidae, Carabidae) from the Gorongosa National Park (Mozambique)
The Gorongosa National Park (Mozambique) is one of the most emblematic protected areas in Africa, well known for its vertebrate biodiversity and restoration ecology efforts following the Mozambican civil war in 1992. The invertebrate biodiversity of Gorongosa National Park is still poorly studied, although the scarce information available indicates the existence of a rich number of species, particularly ground-beetles. The study of Caraboidea beetles is key for designing conservation practices since they are frequently used as biodiversity and ecological indicators and provide valuable information to help decision making. Therefore, the diversity assessment of Caraboidea beetles using standardized methodologies, can be used to quantify the effects of climate change in areas identified as vulnerable to climate change, such as the Gorongosa National Park. We report the occurrence of five tiger-beetles (Cicindelidae) and 93 ground-beetles (Carabidae) species/morphospecies in Gorongosa National Park from a field survey funded by the ECOASSESS project. Sampling was performed in the four main habitat types present in the park (miombo tropical forest, mixed dry forest, transition forest and grasslands) between October 25th and November 25th. In this sampling window, the turnover of Caraboidea species from the dry season to the wet season was recorded for the first time. Twenty-eight species of ground-beetles are new records to Mozambique, including 4 new subgenera and 2 new genera. Additional information on species phenology and habitat preferences is also provided.FUNDING: This study was supported by the Project ECOASSESS – A biodiveristy and ECOlogical ASSESSment of soil fauna of Gorongosa National Park (Mozambique) (PTDC/BIA-CBI/29672/2017) funded through national funds by FCT / MCTES (PIDDAC) under the Programme All Scientific Domains. Marie Bartz was contracted by the University of Coimbra (contract nr. IT057-19-7955) through financial support by the Project/R&D Instituition ECOASSESS. Sara Mendes was financially supported by FCiências – Associação para a investigação e Desenvolvimento de Ciências through research grants funded by the Project/R&D Institution ECOASSESS. Mário Boieiro and Sérgio Timóteo were supported by FCT under contracts DL57/2016/CP1375/CT0001 and CEECIND/00135/2017, respectively.info:eu-repo/semantics/publishedVersio
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