Detection of Change in Fluorescence Between Reactive Cyan and the Yellow Fluorophores Using a-SiC:H Multilayer Transducers

The transducer consists of a semiconductor device based on two stacked -i-n heterostructures that were designed to detect the emissions of the fluorescence resonance energy transfer between fluorophores in the cyan (470 nm) and yellow (588 nm) range of the spectrum. This research represents a preliminary study on the use of such wavelength-sensitive devices as photodetectors for this kind of application. The device was characterized through optoelectronic measurements concerning spectral response measurements under different electrical and optical biasing conditions. To simulate the fluorescence resonance energy transfer (FRET) pairs, a chromatic time-dependent combination of cyan and yellow wavelengths was applied to the device. The generated photocurrent was measured under reverse and forward bias to read out the output photocurrent signal. A different wavelength-biasing light was also superimposed. Results show that under reverse bias, the photocurrent signal presents four separate levels, each one assigned to the different wavelength combinations of the FRET pairs. If a blue background is superimposed, the yellow channel is enhanced and the cyan suppressed, while under red irradiation, the opposite behavior occurs. So, under suitable biasing light, the transducer is able to detect separately the cyan and yellow fluorescence pairs. An electrical model, supported by a numerical simulation, supports the transduction mechanism of the device.

Detection of FRET signals with a wavelength sensitive device based on a-SiC:H

Glucose sensing is an issue with great interest in medical and biological applications. One possible approach to glucose detection takes advantage of measuring changes in fluorescence resonance energy transfer (FRET) between a fluorescent donor and an acceptor within a protein which undergoes glucose-induced changes in conformation. This demands the detection of fluorescent signals in the visible spectrum. In this paper we analyzed the emission spectrum obtained from fluorescent labels attached to a protein which changes its conformation in the presence of glucose using a commercial spectrofluorometer. Different glucose nanosensors were used to measure the output spectra with fluorescent signals located at the cyan and yellow bands of the spectrum. A new device is presented based on multilayered a-SiC:H heterostructures to detect identical transient visible signals. The transducer consists of a p-i'(a-SiC:H)-n/p-i(a-Si:H)-n heterostructure optimized for the detection of the fluorescence resonance energy transfer between fluorophores with excitation in the violet (400 nm) and emissions in the cyan (470 nm) and yellow (588 nm) range of the spectrum. Results show that the device photocurrent signal measured under reverse bias and using appropriate steady state optical bias, allows the separate detection of the cyan and yellow fluorescence signals. (C) 2013 Elsevier B.V. All rights reserved.

Viability of the use of thin-film A-SiC:H photodiodes for protein identification

In this paper, we present a multilayer device based on a-Si:H/a-SiC:H that operates as photodetector and optical filter. The use of such device in protein detection applications is relevant in Fluorescence Resonance Energy Transfer (FRET) measurements. This method demands the detection of fluorescent signals located at specific wavelengths bands in the visible part of the electromagnetic spectrum. The device operates in the visible range with a selective sensitivity dependent on electrical and optical bias. Several nanosensors were tested with a commercial spectrophotometer to assess the performance of FRET signals using glucose solutions of different concentrations. The proposed device was used to demonstrate the possibility of FRET signals detection, using visible signals of similar wavelength and intensity. The device sensitivity was tuned to enhance the wavelength band of interest using steady state optical bias at 400 nm. Results show the ability of the device to detect signals in this range. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Non-enzymatic assay for glucose by using immobilized whole-cells of E. coli containing glucose binding protein fused to fluorescent proteins

Glucose monitoring in vivo is a crucial issue for gaining new understanding of diabetes. Glucose binding protein (GBP) fused to two fluorescent indicator proteins (FLIP) was used in the present study such as FLIP-glu- 3.2 mM. Recombinant Escherichia coli whole-cells containing genetically encoded nanosensors as well as cell-free extracts were immobilized either on inner epidermis of onion bulb scale or on 96-well microtiter plates in the presence of glutaraldehyde. Glucose monitoring was carried out by Förster Resonance Energy Transfer (FRET) analysis due the cyano and yellow fluorescent proteins (ECFP and EYFP) immobilized in both these supports. The recovery of these immobilized FLIP nanosensors compared with the free whole-cells and cell-free extract was in the range of 50–90%. Moreover, the data revealed that these FLIP nanosensors can be immobilized in such solid supports with retention of their biological activity. Glucose assay was devised by FRET analysis by using these nanosensors in real samples which detected glucose in the linear range of 0–24 mM with a limit of detection of 0.11 mM glucose. On the other hand, storage and operational stability studies revealed that they are very stable and can be re-used several times (i.e. at least 20 times) without any significant loss of FRET signal. To author's knowledge, this is the first report on the use of such immobilization supports for whole-cells and cell-free extract containing FLIP nanosensor for glucose assay. On the other hand, this is a novel and cheap high throughput method for glucose assay.

Homogneous time resolved fluorescence: a new technique for detection and quantifications of infliximab in autoimmune patients

RESUMO:As terapias biológicas revolucionaram o tratamento das doenças autoimunes nos últimos anos. Tipicamente têm como alvos mediadores importantes no mecanismo das doenças. Os antagonistas do fator de necrose tumoral-α (TNF-α) são um grupo de agentes biológicos muito prescrito, pois estão indicados no tratamento de doenças imuno-mediadas comuns, tais como artrite reumatoide, artrite idiopática juvenil, artrite psoriática, espondilite anquilosante, doença de Crohn e colite ulcerosa. Com o uso frequente de inibidores do TNF-α, tem-se tornado evidente que estes agentes têm um potencial imunogénico importante, que pode comprometer o prognóstico a longo prazo dos doentes cronicamente tratados. A produção de anticorpos anti-fármaco parece causar falência terapêutica secundária em muitos doentes. Um dos efeitos dos anticorpos anti-fármaco é o aumento da eliminação do fármaco. A eliminação do fármaco, por sua vez, varia entre indivíduos, refletindo diferentes perfis farmacocinéticos. A determinação dos níveis séricos mínimos do agente anti-TNF-α é assim muito informativa e pode auxiliar nas decisões terapêuticas. Contudo, os testes imunológicos para determinar as concentrações séricas do fármaco não estão facilmente disponíveis na prática clínica. De forma a investigar uma nova técnica potencialmente fidedigna e prática para a deteção e quantificação dos agentes biológicos anti-TNF-α, foi testada a técnica por HTRF (homogeneous time-resolved fluorescence resonance energy transfer) para a determinação de concentrações séricas de infliximab. Apesar de apresentar algumas limitações relacionadas com as condições de leitura da fluorescência, esta técnica provou obter resultados próximos das concentrações obtidas por ELISA (enzyme-linked immunosorbent assay) bridging. Adicionalmente, tem a vantagem de ser de execução muito mais fácil e rápida. Deste modo, a técnica por HTRF poderá ser otimizada e tornar-se uma valiosa ferramenta laboratorial para orientar as decisões terapêuticas em doentes autoimunes com falência da terapêutica anti-TNF-α.--------- ABSTRACT: Biologic therapies revolutionized the treatment of autoimmune diseases in the last years. Typically, they target important disease mediators. Tumor necrosis factor-alpha (TNF-α) antagonists constitute a very prescribed group of biologic agents as they are indicated for the treatment of common immune-mediated diseases, such as rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn’s disease and ulcerative colitis. With the increasing use of TNF-α inhibitors it has been noticed that they have an important immunogenic potential that can compromise long-term outcomes in chronically treated patients. The production of anti-drug antibodies seems to cause secondary therapeutic failure in many patients. One of the effects of anti-drug antibodies is the enhancement of drug clearance. Drug clearance, in turn, varies among individuals, reflecting different pharmacokinetic profiles. Determination of serum anti-TNF-α drug trough levels is though very informative and could support treatment decisions. However, immunologic assays to determine drug serum concentrations are not readily available in clinical practice. In order to investigate a potentially reliable and practical new technique for detection and quantification of anti-TNF-α biologic agents, homogeneous time-resolved fluorescence resonance energy transfer (HTRF) technique was tested for determination of serum infliximab concentrations. Although presenting some limitations related with fluorescence reading conditions, this technique proved to give results close to the concentrations obtained by the widely used bridging enzyme-linked immunosorbent assay (ELISA). In addition, it has the advantage of being much easier and faster to perform. Thus, HTRF technique can be optimized and become a valuable laboratorial tool to guide treatment decisions in autoimmune patients with anti-TNF-α therapy failure.

Terahertz response of patterned epitaxial graphene

We study the interaction between polarized terahertz (THz) radiation and micro-structured large-area graphene in transmission geometry. In order to efficiently couple the radiation into the two-dimensional material, a lateral periodic patterning of a closed graphene sheet by intercalation doping into stripes is chosen. We observe unequal transmittance of the radiation polarized parallel and perpendicular to the stripes. The relative contrast, partly enhanced by Fabry-Perot oscillations reaches 20 %. The effect even increases up to 50 % when removing graphene stripes in analogy to a wire grid polarizer. The polarization dependence is analyzed in a large frequency range from < 80 GHz to 3 THz, including the plasmon-polariton resonance. The results are in excellent agreement with theoretical calculations based on the electronic energy spectrum of graphene and the electrodynamics of the patterned structure

Effect of surface plasmon resonance in TiO2/Au thin films on the fluorescence of self-assembled CdTe QDs structure

The exceptional properties of localised surface plasmons (LSPs), such as local field enhancement and confinement effects, resonant behavior, make them ideal candidates to control the emission of luminescent nanoparticles. In the present work, we investigated the LSP effect on the steady-state and time-resolved emission properties of quantum dots (QDs) by organizing the dots into self-assembled dendrite structures deposited on plasmonic nanostructures. Self-assembled structures consisting of water-soluble CdTe mono-size QDs, were developed on the surface of co-sputtered TiO2 thin films doped with Au nanoparticles (NPs) annealed at different temperatures. Their steady-state fluorescence properties were probed by scanning the spatially resolved emission spectra and the energy transfer processes were investigated by the fluorescence lifetime imaging (FLIM) microscopy. Our results indicate that a resonant coupling between excitons confined in QDs and LSPs in Au NPs located beneath the self-assembled structure indeed takes place and results in (i) a shift of the ground state luminescence towards higher energies and onset of emission from excited states in QDs, and (ii) a decrease of the ground state exciton lifetime (fluorescence quenching).

Transferência de Energia sem fios para carregamento de baterias

Dissertação de mestrado integrado em Engenharia Eletrónica Industrial e Computadores

Magnetoliposomes based on manganese ferrite nanoparticles as nanocarriers for antitumor drugs

Manganese ferrite nanoparticles with a size distribution of 26 ± 7 nm (from TEM measurements) were synthesized by the coprecipitation method. The obtained nanoparticles exhibit a superparamagnetic behaviour at room temperature with a magnetic squareness of 0.016 and a coercivity field of 6.3 Oe. These nanoparticles were either entrapped in liposomes (aqueous magnetoliposomes, AMLs) or covered with a lipid bilayer, forming solid magnetoliposomes (SMLs). Both types of magnetoliposomes, exhibiting sizes below or around 150 nm, were found to be suitable for biomedical applications. Membrane fusion between magnetoliposomes (both AMLS and SMLs) and GUVs (giant unilamellar vesicles), the latter used as models of cell membranes, was confirmed by F¨orster Resonance Energy Transfer (FRET) assays, using a NBD labeled lipid as the energy donor and Nile Red or rhodamine B-DOPE as the energy acceptor. A potential antitumor thienopyridine derivative was successfully incorporated into both aqueous and solid magnetoliposomes, pointing to a promising application of these systems in oncological therapy, simultaneously as hyperthermia agents and nanocarriers for antitumor drugs.

Insights into molecular mechanisms regulating the activity of multidomain proteins in living cells using FRET-FLIM

FRET-FLIM, ENERGY TRANSFER, LIFETIME, DECAY ASSOCIATED SPECTRUM, DAS, KINASE, MAGUKS, SINGLE PHOTON COUNTING, PICOSECOND-TIME RESOLVED FLUORESCENCE SPECTROSCOPY, GFP, CFP, YFP, TOPAZ, NANOMETER, MICROSCOPY, LYMPHOCYTES, LCK, SAP97

Oligomerization of the alpha 1a- and alpha 1b-adrenergic receptor subtypes. Potential implications in receptor internalization.

We combined biophysical, biochemical, and pharmacological approaches to investigate the ability of the alpha 1a- and alpha 1b-adrenergic receptor (AR) subtypes to form homo- and hetero-oligomers. Receptors tagged with different epitopes (hemagglutinin and Myc) or fluorescent proteins (cyan and green fluorescent proteins) were transiently expressed in HEK-293 cells either individually or in different combinations. Fluorescence resonance energy transfer measurements provided evidence that both the alpha 1a- and alpha 1b-AR can form homo-oligomers with similar transfer efficiency of approximately 0.10. Hetero-oligomers could also be observed between the alpha 1b- and the alpha 1a-AR subtypes but not between the alpha 1b-AR and the beta2-AR, the NK1 tachykinin, or the CCR5 chemokine receptors. Oligomerization of the alpha 1b-AR did not require the integrity of its C-tail, of two glycophorin motifs, or of the N-linked glycosylation sites at its N terminus. In contrast, helix I and, to a lesser extent, helix VII were found to play a role in the alpha 1b-AR homo-oligomerization. Receptor oligomerization was not influenced by the agonist epinephrine or by the inverse agonist prazosin. A constitutively active (A293E) as well as a signaling-deficient (R143E) mutant displayed oligomerization features similar to those of the wild type alpha 1b-AR. Confocal imaging revealed that oligomerization of the alpha1-AR subtypes correlated with their ability to co-internalize upon exposure to the agonist. The alpha 1a-selective agonist oxymetazoline induced the co-internalization of the alpha 1a- and alpha 1b-AR, whereas the alpha 1b-AR could not co-internalize with the NK1 tachykinin or CCR5 chemokine receptors. Oligomerization might therefore represent an additional mechanism regulating the physiological responses mediated by the alpha 1a- and alpha 1b-AR subtypes.

Nano-electro-mechanical in dynamical cellular processes

Report for the scientific sojourn carried out at the Department of Structure and Constituents of Matter during 2007.The main focus of the work was on phenomena related to nano-electromechanical processes that take place on a cellular level. Additionally, it has also been performed independent work related to charge and energy transfer in bio molecules, energy transfer in coupled spin systems as well as electrodynamics of nonlinear metamaterials.

Ligand binding transmits conformational changes across the membrane-spanning region to the intracellular side of the 5-HT3 serotonin receptor.

Conformational changes of channel activation: Five enhanced green fluorescent protein (EGFP) molecules (green cylinders) were integrated into the intracellular part of the homopentameric ionotropic 5-HT3 receptor. This allowed the detection of extracellular binding of fluorescent ligands (?) to EGFP by FRET, and also enabled the quantification of agonist-induced conformational changes in the intracellular region of the receptor by homo-FRET between EGFPs. The approach opens novel ways for probing receptor activation and functional screening of therapeutic compounds.

Fluorescence imaging reveals the nuclear behavior of peroxisome proliferator-activated receptor/retinoid X receptor heterodimers in the absence and presence of ligand.

In a global approach combining fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), and fluorescence resonance energy transfer (FRET), we address the behavior in living cells of the peroxisome proliferator-activated receptors (PPARs), a family of nuclear receptors involved in lipid and glucose metabolism, inflammation control, and wound healing. We first demonstrate that unlike several other nuclear receptors, PPARs do not form speckles upon ligand activation. The subnuclear structures that may be observed under some experimental conditions result from overexpression of the protein and our immunolabeling experiments suggest that these structures are subjected to degradation by the proteasome. Interestingly and in contrast to a general assumption, PPARs readily heterodimerize with retinoid X receptor (RXR) in the absence of ligand in living cells. PPAR diffusion coefficients indicate that all the receptors are engaged in complexes of very high molecular masses and/or interact with relatively immobile nuclear components. PPARs are not immobilized by ligand binding. However, they exhibit a ligand-induced reduction of mobility, probably due to enhanced interactions with cofactors and/or chromatin. Our study draws attention to the limitations and pitfalls of fluorescent chimera imaging and demonstrates the usefulness of the combination of FCS, FRAP, and FRET to assess the behavior of nuclear receptors and their mode of action in living cells.

Molecular detection of Schistosoma japonicum in infected snails and mouse faeces using a real-time PCR assay with FRET hybridisation probes

A real-time polymerase chain reaction (PCR) assay with fluorescence resonance energy transfer (FRET) hybridisation probes combined with melting curve analysis was developed to detect Schistosoma japonicum in experimentally infected snails and in faecal samples of infected mice. This procedure is based on melting curve analysis of a hybrid between an amplicon from the S. japonicum internal transcribed spacer region 2 sequence, which is a 192-bp S. japonicum-specific sequence, and fluorophore-labelled specific probes. Real-time FRET PCR could detect as little as a single cercaria artificially introduced into a pool of 10 non-infected snails and a single egg inoculated in 100 mg of non-infected mouse faeces. All S. japonicum-infected snails and all faecal samples from infected mice were positive. Non-infected snails, non-infected mouse faeces and genomic DNA from other parasites were negative. This assay is rapid and has potential for epidemiological S. japonicum surveys in snails, intermediate hosts and faecal samples of final hosts.