Browsing by Author "Paulo, Pedro M. R."
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- Fluorescent dye nano-assemblies by thiol attachment directed to the tips of gold nanorods for effective emission enhancementPublication . Botequim, David; Silva, Inês I. R.; Serra, Sofia G.; EP, Melo; Prazeres, Duarte M. F.; Costa, Sílvia M. B.; Paulo, Pedro M. R.The conjugation of dye-labelled DNA oligonucleotides with gold nanorods has been widely explored for the development of multifunctional fluorescent nanoprobes. Here, we show that the functionalization route is crucial to achieve enhanced emission in dye nano-assemblies based on gold nanorods. By using a tip-selective approach for thiol attachment of dye molecules onto gold nanorods, it was possible to effectively increase the emission by more than 10-fold relatively to that of a free dye. On the other hand, a non-selective approach revealed that indiscriminate surface functionalization has a detrimental effect on the enhancement. Simulations of discrete dipole approximation gave further insight into the surface distribution of plasmon-enhanced emission by confirming that tip regions afford an effective enhancement, while side regions exhibit a negligible effect or even emission quenching. The contrast between dye nano-assemblies obtained from tip- and non-selective functionalization was further characterized by single-particle fluorescence emission. These studies showed that tip-functionalized gold nanorods with an average of only 30 dye molecules have a comparable to or even stronger emission than non-selectively functionalized particles with approximately 10 times more dye molecules. The results herein reported could significantly improve the performance of dye nano-assemblies for imaging or sensing applications.
- Gold nanorods functionalized with DNA oligonucleotide probes for biosensing and plasmon-enhanced fluorescence detectionPublication . Alughare, Zohre Eskandari; Paulo, Pedro M. R.; Garcia, Ana RosaGold 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.
- Live-cell FRET imaging reveals clustering of the prion protein at the cell surface induced by infectious prionsPublication . Tavares, Evandro; Macedo, J.A.; Paulo, Pedro M. R.; Sousa, Catarina; Lopes, Carlos; Melo, EduardoPrion 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.
- Stress-induced protein disaggregation in the endoplasmic reticulum catalysed by BiPPublication . Melo, Eduardo; Konno, Tasuku; Farace, Ilaria; Awadelkareem, Mosab Ali; Skov, Lise R.; Teodoro, Fernando; Sancho, Teresa; Paton, Adrienne W.; Paton, James C.; Fares, Matthew; Paulo, Pedro M. R.; Zhang, Xin; Avezov, EdwardProtein synthesis is supported by cellular machineries that ensure polypeptides fold to their native conformation, whilst eliminating misfolded, aggregation prone species. Protein aggregation underlies pathologies including neurodegeneration. Aggregates' formation is antagonised by molecular chaperones, with cytoplasmic machinery resolving insoluble protein aggregates. However, it is unknown whether an analogous disaggregation system exists in the Endoplasmic Reticulum (ER) where -30% of the proteome is synthesised. Here we show that the ER of a variety of mammalian cell types, including neurons, is endowed with the capability to resolve protein aggregates under stress. Utilising a purpose-developed protein aggregation probing system with a sub-organellar resolution, we observe steady-state aggregate accumulation in the ER. Pharmacological induction of ER stress does not augment aggregates, but rather stimulate their clearance within hours. We show that this dis-sagregation activity is catalysed by the stress-responsive ER molecular chaperone - BiP. This work reveals a hitherto unknow, non-redundant strand of the proteostasis-restorative ER stress response.