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Characterization of white light emitting CdSe quantum dots2014 August 1900 (has links)
A novel type of white light emitting semiconductor quantum dot was characterized at the ensemble and single-molecule level. This kind of semiconductor nanocrystal can be made into white light emitting diodes, which have the potential to replace conventional lighting sources. The quantum dots used in this thesis consisted of a cadmium selenide (CdSe) core, capped with ZnS, and have a surface polymer coating of poly(acrylic acid) (PAA). We have characterized the quantum dot size distribution by using dynamic light scattering (DLS), transmission electron microscopy (TEM), atomic force microscopy (AFM) and UV-Vis spectroscopy. Based on these measurements, it is clear that the white quantum dots are polydisperse, with a core size of 2.4 ± 0.5 nm, though the polymer coating swells considerably in aqueous solution. In order to explore the optical properties, the absorption and emission spectra of the ensemble quantum dots solution were measured and compared to “standard” commercial quantum dots. The emission spectrum of the white quantum dots showed two peaks, a strong blue emission peak and a weaker red emission peak. The fluorescence quantum yield of the white quantum dots was found to be less than that of commercial quantum dots. To explore the behavior of individual quantum dots, spatially-resolved single-molecule images were obtained by a dual-view single molecule fluorescence microscopy with a beam splitter which can separate the emission into red and blue components. It was found that individual white CdSe nanocrystals have a broad emission spectrum and the samples did not consist of a mixed population of red emitters and blue emitters. These results suggest that these white light emitting quantum dots can be used for pure white light LEDs and are a good candidate for the replacement for conventional lighting sources.
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Synthesis and preliminary study of a cryptand designed to allow potassium ion displacement of a fluorescence quencherYao, Min January 1988 (has links)
A new cryptand, 1,18-bis[(phenylmethoxy)methyl]-3,16,20,23,26,29,32,35-octaoxatetracyclo-[16.9.9,05,10 09,14]hexatriaconta-5,7,9,11,13-pentaene was synthesized from cis-9,19-bis(benzyloxymethyl)-1,4,7,11,14,17-hexaoxacycloeicosane-9,l9-diyldimethanol and l,5-bis(bromomethyl)naphthlene in the presence of t-butoxide base. The overall syntheses involved five steps. The new cryptand compound has a melting point at 164-1650 C, and the yield was 18.5%. The NMR spectra and the crystal structure determination proved the synthesized cryptand the same as that desinged.Fluorescent measurements were performed with this cryptand. The fluorescence was found to be quenched by cesium ion, and the quenching increased with the increasing of cesium ion concentration. The average quenching of the fully complexed (with Cs +) cryptand was estimated as 20 + 2% by a computerized data fitting program. The complexation constant was determined to be 16 ± 5 M-1. A possible explanation for these observations is presented. / Department of Chemistry
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Fluorescent polycyclic ligands : strategies towards the synthesis and evaluation of fluorescently labelled receptor and enzyme ligands / Jacques JoubertJoubert, Jacques January 2012 (has links)
Neurodegenerative disorders, including Alzheimer's and Parkinson's disease, and the
development of neuroprotective agents have received significant research attention in recent
years. Development of novel imaging techniques to study the biological mechanisms involved
in the progression of these disorders have become an area of research interest. The design of
novel small molecule imaging probes in combination with modem imaging techniques may
provide information on neuroprotective binding site• interactions and would assist in the
design of novel biological assay methods. Techniques to visualize physiological or
pathophysiological changes in proteins and living cells have become increasingly important in
biomedical sciences, especially fluorescent techniques. Fluorescent ligands in combination
with sophisticated fluorescent imaging technologies are useful tools to analyze and clarify the
roles of biomolecules in living cells, affording high spatial and temporal resolution.
This study is based on the development of polycyclic fluorescent ligands, which may be used
in the study of receptor-ligand and/or enzyme-ligand interactions, utilizing these fluorescently
labeled ligands in combination with fluorescent imaging techniques. Fluorescent conjugates
with high affinity for the• N-methyl-D-aspartate (NMDA) receptor, voltage gated calcium
channels (VGCC) and/or the nitric oxide synthase (NOS) enzyme were designed and
synthesised with the aim to directly measure binding of these novel molecules to receptors
and/or enzymes.
The first goal was to develop fluorescent ligands that exhibit similar inhibitory activity on
NOS compared to the well-known selective neuronal NOS inhibitor 7-nitroindazole (7-NI).
Polycyclic compounds, including amantadine and pentacycloundecane derivatives, were
conjugated to fluorescent moieties that resemble the structure of 7-NI. It was thought that the
lipophilic nature of the polycyclic compounds would increase the activity of the fluorescent
moieties by facilitating increased blood brain barrier permeability and penetration through cell
membranes. This would also potentially increase the selectivity of the novel conjugated
compounds as selective neuronal NOS inhibitors, similar to 7-NI. The results from the NOS
inhibition studies indicated that the novel fluorescent conjugates (5-14) inhibited the NOS
enzyme at micromolar concentrations. Although none of the novel fluorescent polycyclic
compounds were found to be more potent than 7-NI (IC50 = 0.11 11M), the indazole
pentacyclorindecane (5), the coumarin-adamantane (7), the dansyl-adamantane (8), and the
cyanoisoindole-adamantane (11) conjugates, exhibited IC5o values below 1 uM. These
compounds could possibly be used as molecular probes in the development of high-throughput
screening or competitive NOS displacement assays. Further studies on isoform
selectivity will elaborate on the potential of these compounds as fluorescent molecular probes.
The aforementioned fluorescent derivatives were further developed resulting in a series of
novel fluorescent polycyclic conjugates with potent NOS inhibition indicating the potential of
these compounds as neuroprotective agents. Due to the polycyclic structure's inherent
inhibitory activity towards the NMDA receptor and VGCC we evaluated these derivatives as
possible multifunctional neuroprotective agents acting on various neuroprotective targets. In
the biological studies it was observed that four adamantane fluorescent compounds (7, 8, 10,
11) exhibited a high degree of inhibitory activity against the NOS enzyme and NMDA
receptor and blocked VGCC. The fluorescent compounds were further able to scavange
detrimental neurodegenerative free radicals. In silica studies also predicted a high degree of
oral bioavailability and that these novel compounds should be effectively transported across
the blood brain barrier.
Taking the positive findings on the inhibition of the NMDA receptor and VGCC activity of
the novel fluorescent polycyclic ligands into account we focused on the expansion of this
series. This resulted in the synthesis of a series of fluorescent derivatives utilizing
adamantane-3-aminopropanol as an intermediate to extend the chain length between the
adamantyl and fluorescent moieties, to potentially reduce sterical hindrance and increase
activity. These novel adamantane-3-aminopropanol fluorescent ligands were also evaluated
for inhibition of the NMDA receptor and VGCC. The coumarin-, dansyl- and cyanoisoindole
adamantane-3-aminopropanol fluorescent conjugates (15, 16, 19) displayed significant VGCC
inhibition, with the dansyl (16) and di-nitrobenzene (20) fluorescent derivatives exhibiting
NMDA receptor antagonistic activity. All these compounds showed improved activity when
compared to known NMDA receptor and VGCC inhibitors in this class. Generally it was
observed that the increased chain length analogues had improved VGCC inhibition and
NMDA receptor activity when compared to their directly• conjugated counterparts. This led to
the conclusion that an increase in chain length might indicate deeper immersion into the
NMDA receptor and VGCC which may be necessary for stronger interaction with their
putative binding sites. The dansyl analogue, N-[3-(1-adamantylamino)propyl]-5-
dimethylaminonaphthalene-1-sulfonamide (16), was further used as a fluorescent NMDA
receptor ligand in a fluorescent competition assay, utilizing known NMDA receptor inhibitors
to demonstrate the possible applications of these novel fluorescent analogues and their benefit
over the use of hazardous and expensive radioligand binding studies.
Further investigation on the application of these derivatives, especially on the NOS enzyme
and the NMDA receptor, will develop their potential as fluorescent ligands in the study of
neurodegeneration and may also yield novel therapeutic agents against neurodegenerative
disorders. / PhD (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2012
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844 |
Fluorescent polycyclic ligands : strategies towards the synthesis and evaluation of fluorescently labelled receptor and enzyme ligands / Jacques JoubertJoubert, Jacques January 2012 (has links)
Neurodegenerative disorders, including Alzheimer's and Parkinson's disease, and the
development of neuroprotective agents have received significant research attention in recent
years. Development of novel imaging techniques to study the biological mechanisms involved
in the progression of these disorders have become an area of research interest. The design of
novel small molecule imaging probes in combination with modem imaging techniques may
provide information on neuroprotective binding site• interactions and would assist in the
design of novel biological assay methods. Techniques to visualize physiological or
pathophysiological changes in proteins and living cells have become increasingly important in
biomedical sciences, especially fluorescent techniques. Fluorescent ligands in combination
with sophisticated fluorescent imaging technologies are useful tools to analyze and clarify the
roles of biomolecules in living cells, affording high spatial and temporal resolution.
This study is based on the development of polycyclic fluorescent ligands, which may be used
in the study of receptor-ligand and/or enzyme-ligand interactions, utilizing these fluorescently
labeled ligands in combination with fluorescent imaging techniques. Fluorescent conjugates
with high affinity for the• N-methyl-D-aspartate (NMDA) receptor, voltage gated calcium
channels (VGCC) and/or the nitric oxide synthase (NOS) enzyme were designed and
synthesised with the aim to directly measure binding of these novel molecules to receptors
and/or enzymes.
The first goal was to develop fluorescent ligands that exhibit similar inhibitory activity on
NOS compared to the well-known selective neuronal NOS inhibitor 7-nitroindazole (7-NI).
Polycyclic compounds, including amantadine and pentacycloundecane derivatives, were
conjugated to fluorescent moieties that resemble the structure of 7-NI. It was thought that the
lipophilic nature of the polycyclic compounds would increase the activity of the fluorescent
moieties by facilitating increased blood brain barrier permeability and penetration through cell
membranes. This would also potentially increase the selectivity of the novel conjugated
compounds as selective neuronal NOS inhibitors, similar to 7-NI. The results from the NOS
inhibition studies indicated that the novel fluorescent conjugates (5-14) inhibited the NOS
enzyme at micromolar concentrations. Although none of the novel fluorescent polycyclic
compounds were found to be more potent than 7-NI (IC50 = 0.11 11M), the indazole
pentacyclorindecane (5), the coumarin-adamantane (7), the dansyl-adamantane (8), and the
cyanoisoindole-adamantane (11) conjugates, exhibited IC5o values below 1 uM. These
compounds could possibly be used as molecular probes in the development of high-throughput
screening or competitive NOS displacement assays. Further studies on isoform
selectivity will elaborate on the potential of these compounds as fluorescent molecular probes.
The aforementioned fluorescent derivatives were further developed resulting in a series of
novel fluorescent polycyclic conjugates with potent NOS inhibition indicating the potential of
these compounds as neuroprotective agents. Due to the polycyclic structure's inherent
inhibitory activity towards the NMDA receptor and VGCC we evaluated these derivatives as
possible multifunctional neuroprotective agents acting on various neuroprotective targets. In
the biological studies it was observed that four adamantane fluorescent compounds (7, 8, 10,
11) exhibited a high degree of inhibitory activity against the NOS enzyme and NMDA
receptor and blocked VGCC. The fluorescent compounds were further able to scavange
detrimental neurodegenerative free radicals. In silica studies also predicted a high degree of
oral bioavailability and that these novel compounds should be effectively transported across
the blood brain barrier.
Taking the positive findings on the inhibition of the NMDA receptor and VGCC activity of
the novel fluorescent polycyclic ligands into account we focused on the expansion of this
series. This resulted in the synthesis of a series of fluorescent derivatives utilizing
adamantane-3-aminopropanol as an intermediate to extend the chain length between the
adamantyl and fluorescent moieties, to potentially reduce sterical hindrance and increase
activity. These novel adamantane-3-aminopropanol fluorescent ligands were also evaluated
for inhibition of the NMDA receptor and VGCC. The coumarin-, dansyl- and cyanoisoindole
adamantane-3-aminopropanol fluorescent conjugates (15, 16, 19) displayed significant VGCC
inhibition, with the dansyl (16) and di-nitrobenzene (20) fluorescent derivatives exhibiting
NMDA receptor antagonistic activity. All these compounds showed improved activity when
compared to known NMDA receptor and VGCC inhibitors in this class. Generally it was
observed that the increased chain length analogues had improved VGCC inhibition and
NMDA receptor activity when compared to their directly• conjugated counterparts. This led to
the conclusion that an increase in chain length might indicate deeper immersion into the
NMDA receptor and VGCC which may be necessary for stronger interaction with their
putative binding sites. The dansyl analogue, N-[3-(1-adamantylamino)propyl]-5-
dimethylaminonaphthalene-1-sulfonamide (16), was further used as a fluorescent NMDA
receptor ligand in a fluorescent competition assay, utilizing known NMDA receptor inhibitors
to demonstrate the possible applications of these novel fluorescent analogues and their benefit
over the use of hazardous and expensive radioligand binding studies.
Further investigation on the application of these derivatives, especially on the NOS enzyme
and the NMDA receptor, will develop their potential as fluorescent ligands in the study of
neurodegeneration and may also yield novel therapeutic agents against neurodegenerative
disorders. / PhD (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2012
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Characterization of gemini nanoparticle assembly by fluorescence correlation spectroscopyDong, Chilbert 12 December 2013 (has links)
Research in the field of non-viral gene delivery has demonstrated that a deeper understanding of the fundamental processes of nanoparticle assembly is required in order to improve their efficacy. While gemini nanoparticles (gemini NPs) and other non-viral delivery systems have been vigorously characterized using several techniques, our knowledge is still incomplete. The first objective of this study was the development of new methodology using fluorescence correlation spectroscopy (FCS) to investigate the stages of gemini NPs assembly. It was demonstrated that by labeling the plasmid, different stages of gemini NP assembly from the gemini-plasmid pre-complex (GP) to the final gemini nanoparticle (or gemini-plasmid-lipid complex; GPL), could be studied. Based on diffusion coefficients and particle numbers extrapolated from the autocorrelation function (ACF), FCS was able to determine that each phase of assembly had distinct characteristics. The FCS study using 12-3-12 gemini surfactant showed that both the diffusion coefficient and particle number of GPs (0.98??0.31 x 10-12 m2/s) was significantly lower than the final GPL (3.11??0.41 x 10-12 m2/s). Based on the Stokes-Einstein equation the particle size was calculated to be 300-500 nm for GP and 200-300 nm for GPLs. The raw intensity histograms showed that both GPs and GPLs are composed of multiple plasmids. Furthermore the study showed that the final GPLs contain fewer plasmids compared to the intermediate GP. FCS results were validated by using existing characterization methods including dynamic light scattering (DLS), zeta potential and dye exclusion assays. The second objective involved the detailed characterization of gemini NP. Nine different gemini surfactants and two different phospholipids were used in a systematic study to assess the effect of gemini surfactant and lipid structure on the final morphology of gemini NP. The study revealed that gemini surfactant structure had a strong effect on structure of GP intermediates, but addition of phospholipids resulted in the formation of uniform gemini NPs. Based on the results of this study a new model for GP and GPL assembly is proposed based on the formation of supramolecular aggregates of gemini-plasmids, governed by gemini surfactant chemical structure, and dispersed by phospholipids to form GPLs.
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Characterization of Starch Nanoparticles by Fluorescence TechniquesYi, Wei 21 May 2015 (has links)
Abstract
The properties of starch nanoparticles (SNPs) labeled with the fluorescent dye pyrene (Py-SNPs) were probed by using fluorescence quenching, pyrene excimer formation, and transmission electron microscopy (TEM). Pyrene labeling of the SNPs was achieved by reacting 1-pyrenebutyric acid with the hydroxyl groups of the SNPs under basic conditions and in the presence of diisopropylcarbodiimide. This procedure did not degrade the SNPs as confirmed by dynamic light scattering (DLS) and afforded a means to generate a pyrene labeling level ranging from 0.5 to 5.0 mol% of the glucose units making up the SNPs. A polymeric quencher was also synthesized to probe the accessibility of the interior of the Py-SNPs by using fluorescence quenching measurements. The polymeric quencher was a 2K poly(ethylene glycol) terminated at one end with a methyl group and a nitropropane group at the other. Unfortunately these quenching experiments were abandoned when it was found that the polymeric quencher synthesized for these experiments absorbed too strongly where pyrene absorbs. Intramolecular pyrene excimer formation in the Py-SNPs was investigated by steady-state and time-resolved fluorescence. These experiments demonstrated that the Py-SNPs contract but do not overlap like linear polymers do in the semi-dilute regime. They also showed that despite the inherent rigidity of starch, the Py-SNPs deformed in water to allow their hydrophobic pyrene labels to cluster toward the center of the SNPs to minimize pyrene-solvent contacts. This segregation of the hydrophobic pyrene labels led to a distinct core-shell structure for the Py-SNPs which was illustrated in TEM images acquired on films prepared with the Py-SNPs. In summary, this thesis has uncovered some unexpected properties of the SNPs. Their branched structure makes their interpenetration difficult in the semi-dilute regime which forces them to contract. SNPs are thus deformable and their deformation can be probed quantitatively by using fluorescence and TEM.
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Fluorescent and Photocaged Lipids to Probe the Ceramide-mediated Reorganization of Biological MembranesCarter Ramirez, Daniel Marcelo 23 January 2013 (has links)
This thesis describes the development of novel fluorescent and photocaged lipids, and their application as tools to probe the morphological effects of ceramide (Cer)-mediated membrane reorganization in supported lipid bilayers. Cer is a sphingolipid found in eukaryotic cells that plays a key role in regulating biological processes such as apoptosis, cell-to-cell communication, differentiation and some types of pathogenesis. Sphingolipid and cholesterol-rich lipid rafts in the plasma membrane are thought to be the point of origin for many of this lipid second messenger’s effects. Cer is formed in the exoplasmic leaflet of the plasma membrane via the enzymatic hydrolysis of sphingomyelin. The compositional complexity of biological membranes has prompted the adoption of simpler model systems to study the effects of Cer generation. When it is directly incorporated into model membranes, Cer segregates into highly ordered domains with physical properties that are distinct from those of the surrounding fluid environments. However, enzymatic generation of Cer induces complex and dynamic membrane heterogeneity that is difficult to interpret and reconcile with its direct incorporation. Here I describe the synthesis of 4-nitrobenzo-2-oxa-1,3-diazol-7-yl (NBD)-labelled cholesterol (Chol) and Cer analogs, and their use as probes in model membranes exhibiting liquid-disordered (Ld) and liquid-ordered (Lo) phase coexistence. The Chol probes reproduce the modest enrichment of Chol in Lo membrane domains as well as the Cer-induced displacement of cholesterol. One of the NBD Chol probes is used to provide direct visualization of Chol redistribution during enzymatic Cer generation, and assists in identifying new features as Cer-rich regions. The NBD-labelled Cer quantifies membrane order using orientational order parameter measurements derived from polarized total internal reflection fluorescence microscopy (pTIRFM) images. The probe reports on changes in membrane order upon enzymatic generation of Cer, and indicates a significant increase in the molecular order of Ld membrane regions that is consistent with the redistribution of Chol into these areas. The probe also identifies de novo Cer-rich domains as areas of particularly high molecular order. In the final project area, 6-Bromo-7-hydroxycoumarin-4-ylmethyl (Bhc)-caged Cers are shown to release Cer rapidly and efficiently upon irradiation with near-visible UV light. The caged lipids are then incorporated into supported membranes and photolyzed to release Cer with a high degree of spatial and temporal control. Controlled Cer generation is then used to drive protein-ganglioside clustering in lipid bilayers.
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Mapping RNA Binding Surfaces on Hfq Using Tryptophan Fluorescence QuenchingHoff, Kirsten E. January 2013 (has links)
<p>Abstract</p><p> Hfq is a pleiotropic posttranscriptional regulator and RNA chaperone that facilitates annealing of trans-encoded sRNA/mRNA pairs. It regulates many different cellular pathways including environmental stress responses, quorum sensing, virulence and maintenance of membrane integrity. Hfq is a member of the Sm/LSm family and forms a homohexamer that has two faces, termed proximal and distal. Hfq preferentially binds A/U rich regions that are near stem loop structures. Crystal structures have shown that poly-A sequences tend to bind the distal face while poly-U sequences bind the proximal face. Currently crystal structures reveal the binding mechanisms for short RNA sequences however; physiologically relevant RNA sequences are typically longer and more structured. To study how these more complex RNA sequences interact with Hfq, a tryptophan fluorescence quenching (TFQ) assay has been developed. Here it is presented that TFQ can correctly identify the binding face for two control sequences, A15 and U6, using the E. coli, S. aureus and L. monocytogenes Hfq homologues. Using fluorescence anisotropy and crystallography it is observed that Trp mutants necessary for TFQ may affect binding to some degree but do not affect the overall structure or RNA binding function of Hfq. TFQ is then used to examine the distal face binding motifs for both Gram-negative (E. coli) and Gram-positive (S. aureus/L. monocytogenes) Hfq, (A-R-N)n and (R-L)n respectively. Using sequences that either fulfilled just (A-R-N)n or both (A-R-N)n and (A-A-N)n motifs it is shown that the distal face motif for Gram-negative Hfq is the more specific (A-A-N)n motif. Using sequences that either fulfilled just (R-L)n or both (R-L)n and (A-L)n motifs it is shown that the Gram-positive distal face motif can be redefined to the (A-L)n motif. Finally TFQ is used to explore autoregulation of E. coli hfq. Two identified binding sites located in the 5'UTR of hfq mRNA, site A and site B, were used for TFQ, along with a longer RNA sequence that contains both sites and their native linker, 5' UTR. TFQ illustrates that the individual sites and the 5' UTR are capable of binding both faces. Each site appears to prefer binding to one face over the other, suggesting a model for hfq 5' UTR mRNA binding to Hfq where either one or two hfq mRNA bind a single Hfq hexamer. In conclusion, TFQ is a straightforward method for analyzing how RNA sequences interact with Hfq that can be utilized to study how longer, physiologically relevant RNA sequences bind Hfq.</p> / Dissertation
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Laser induced fluorescence measurements in inductivity coupled of processing plasmasGomez, S. January 2001 (has links)
No description available.
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Toward the Development of Nucleic Acid Assays Using Fluorescence Resonance Energy Transfer (FRET) and a Novel Label Free Molecular Switching ConstructMassey, Melissa 06 December 2012 (has links)
The research presented in this thesis introduces design criteria for development of a new type of self-contained optical biosensor. The study begins with evaluation of a dual label, fluorescence resonance energy transfer (FRET) bioassay format, and then goes on to demonstrate a signalling platform that uses an immobilized fluorescent intercalating dye so as to avoid labelling of both the target and probe strands.
An extensive survey of FRET pairs that can be used to monitor hybridization events in solution and at solid interfaces was conducted in solution to provide a set of calculated Förster distances for the extrinsic labels Cyanine 3 (Cy3), Cyanine 5 (Cy5), Carboxytetramethylrhodamine (TAMRA), Iowa Black Fluorescence Quencher (IabFQ) and Iowa Black RQ (IabRQ). FRET parameters using thiazole orange (TO) intercalating dye as a FRET donor for various acceptor dye-labelled DNA conjugates in solution were determined. Limitations associated with quenching mechanisms other than those mediated by FRET motivated the development of a molecular switch that contained intercalating dye.
The four binding sites associated with Neutravidin served for assembly of the switch using biotin interactions. One binding site was used to immobilize an unlabelled oligonucleotide probe. The adjacent site was used to immobilize a novel biotinylated TO derivative that could physically reach the probe. On hybridization of the probe with target, the intercalating dye was captured by the hybrid, leading to a change of fluorescence. This reversible signalling mechanism offers a method without nucleic acid labelling to detect nucleic acid association at an interface. A SNP discrimination strategy involving TO and formamide was investigated, and SNP discrimination without the requirement of thermal denaturation was achieved for multiple target lengths, including a 141-base pair PCR amplicon in solution. It was determined that formamide could also provide improvements of signal-to-noise when using thiazole orange to detect hybridization.
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