Polymethine Dye as Sensors of NH 3

We have investigated the properties of polymethine dyes (PMD) and the purpose of using them as sensors of hazardous gases presence. Research indicates that in case of utilizing PMD as a sensor of hazardous gases we need to use the monochromatic light which wavelength corresponds to the inflection point of the long-wave absorption edge of PMD. Such sensor of hazardous gases can detect changes in the optical density of the sensor layer or the light intensity transmitted through the layer

Towards Novel Optical Sensors

This research presented in this thesis is based in the development of optical sensors. More specifically, this project outlines two novel systems that could possible used within this field. The first system is based on a functionalized ruthenium(II) polypyridyl complex and the second is based on derivatives of merocyanine 540. For the first system the synthesis and detailed photophysical properties of a bis(N-methylene-4-pyridinium-4'-pyridine) functionalized ruthenium(II) polypyridyl complex (C2) in solvents of varying polarity and Gutmann's donor number (DN) are discussed. The emission spectrum of (C2) in varying solvents was also simulated and parameters including the Huang-Rhys (Sm) obtained. Respectable correlations of the optical properties of (C2) with the solvent polarity factor (Af) and DN were obtained. These results were used to calculate a difference in dipole moment between ground and excited state of ca 10 D, and propose a simplified potential-energy surface model to explain the results. The excited state lifetime data for (C2) in acetonitrile were collected over a modest temperature range of 60K. The Arrhenius plot of In (kobs) vs 1/T was found to be linear affording a proportionately low activation energy of 5.6 (+/- 0.2) kJ mol-1 when compared to the literature value of 45.5 kJ mol-1 for [Ru(biby)3]2+. For the second system the design of novel merocyanine dyes for use in sensing and photodynamic therapy is given. The parent dye of these molecules being merocyanine 540. With its preferential uptake into leukaemia cells and their subsequent eradication by induced photosensitization, has rewarded it considerable interest over the years in the field of photodynamic therapy. Its ability to act as a photosensitizer depends greatly on its efficiency of intersystem crossing to the triplet manifold. Unfortunately the dominant photoprocess involves photo-isomerization to form a long-lived (unstable) isomer. It is expected that if this isomerization process could be prevented then it would lead to a much more potent photosensitizer. There have been extensive studies into the prevention of this process ranging from structural to electronic changes within the molecule, but sadly to no avail. The design of the derivatives MC1 and MC2 was carried out via the incorporation of a triazamacrocycle into the structure of the molecule. It is predicted that with the binding of a metal to this macrocycle the molecule will be locked into the trans isomer and be unable to undergo photo-isomerization

Near infrared fluorescent sensors for selective glucose recognition

With diabetes becoming a major health concern worldwide, it is growing clear that novel technologies are needed to monitor blood glucose in a manner that is continuous, real-time, and long-term --all while alleviating the burden of pain on the patient. Current meters commonly used for glucose are invasive in nature and require blood samples to offer intermittent readings on blood glucose levels. Despite extensive research on developing minimally invasive or non-invasive ways to monitor blood glucose levels, this objective has remained elusive. Previous attempts in providing a minimally invasive sensor for in vivo blood glucose detection via fluorescence spectroscopy have offered excellent information on how to design and develop a glucose recognition motif while at the same time suffer from a number of limitations such as poor water solubility, stability, and impractical spectroscopic features for in vivo sensing. To realize a viable sensor for long term in vivo monitoring of blood glucose, near IR pH probes based on cyanine fluorophores were designed and synthesized. These probes provided insight into practicality of cyaninebased near IR sensors for use in vivo and served as a starting point to achieve the ultimate goal of glucose recognition. By uniting the methodology for near IR pH sensing and the required architecture for glucose recognition, several NIR glucose sensors based on cyanine platforms were designed, synthesized, and tested

Synthesis and Characterization of Fluorescent Silica Nanoparticles with Large Stokes Shifts for Multiplexed Assays and Imaging

Modern approaches to biological and biomedical analysis demand ever-increasing levels of sensitivity, selectivity, and throughput. This challenge has been addressed in the work described herein via the synthesis, characterization, and proofs of concept of a series of single and multidye copolymerized fluorescent silica nanoparticles with large Stokes shifts and near infrared fluorescence. The prepared fluorescent probes exhibit substantially enhanced fluorescence signals relative to their constituent dyes, good indicators of biocompatibility, and readily distinguishable fluorescence signals, promoting the simultaneous detection of multiple targets and reducing both the cost and time per assay. A novel NIR-fluorescent aminocyanine dye was designed for incorporation into silica nanoparticles, then synthesized and characterized as detailed in the second chapter. Spectroscopic characterization confirmed the intended dye structure and revealed a large Stokes shift, near infrared fluorescence, and a relatively high quantum yield, indicating the suitability of this compound for bioanalytical applications and incorporation into silica nanoparticles as either a standalone fluorophore or as a resonance energy transfer acceptor for other UV-visible dyes. In the third chapter, the incorporation of the novel dye and other commercial dyes into silica nanoparticles is discussed, along with characterization and proofs of concept for in vivo and in vitro applications of the resultant fluorescent labels. Nanoparticle synthetic approaches, dye concentrations, and surface coating densities were optimized for fluorescence intensities and biocompatibility. Nanoparticles containing single and multiple dye species were synthesized per the optimal parameters, producing a series of fluorescent tags with distinct fluorescence signatures and large Stokes shifts. Synthesized nanoparticles were characterized in terms of sizes, synthetic yields, quantum yields, limits of detection, stability, and synthetic reproducibility. The suitability of surface modified nanoparticles to in vitro and in vivo applications was demonstrated via biotin linkage to streptavidin microbeads and fluorescence microscopy imaging, hemocompatibility studies, and protein binding studies. Finally, in the fourth chapter, resonance energy transfer characteristics of multidye copolymerized nanoparticles were characterized. Energy transfer efficiencies between donor-acceptor pairs, numbers of dye molecules per nanoparticle, and average distances between dye molecules were calculated, then Förster radii for different donor-acceptor pairs were estimated using two approaches and compared

Synthesis and Evaluation of Derivatives and Analogs of Xanthene Dyes

Xanthene dyes are one of the oldest synthetic dyes. Fluorescein was first synthesized by von Baeyer in 1871. Since its discovery, it has been extensively studied. Fluorescein can exist as four different ionic forms (cationic form, neutral form, monoanionic form and dianionic form). Under physiological conditions (pH 7.4), fluorescein mainly exists as the dianionic form which grants it large quantum yield and excellent solubility in water. This renders fluorescein and its derivatives useful for studies in biological media. Cysteine (Cys) and homocysteine (Hcy) are of the few amino acids which contain sulfur and both of them are believed to be related to some diseases. Cardiovascular diseases, Alzheimer’s disease and neutral tube defects are reported to be associated with elevated levels of homocysteine in blood plasma. Low levels of cysteine are associated with slowed growth, hair depigmentation, liver damage, etc. The reason for Cys and Hcy being connected with diseases is still unclear. Herein, we designed and synthesized fluorescein-based dyes for detecting Hcy and Cys in the visible spectral region with the highest selectivity. Optical sensors in the near-infrared (NIR) spectral range have captured the attention of researchers interested in studying chemical and biological processes in live tissues because absorption and scattering by endogenous biomolecules are minimal. Three heptamehtine cyanine-based dyes were designed and synthesized for the detection or labeling of Cys and Hcy. Biocompatible hydrogels have been studied in many fields, e.g., drug delivery system, separation processes or sensors (fluorescent polymers). A fluorescein-based hydrogel with potential application for detection of Cys and Hcy were designed and underwent synthesized. Dibenzo-p-dioxin is a structural analog of xanthene but is notorious for the toxicicty of the halogenated members, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Since polyhalogenated dibenzo-p-dioxins (PXDDs) have been shown to be a significant threat to public health, measuring the concentrations of PXDDs in the environment as well as understanding their mechanism of formation and environmental fate is needed. Here we designed a novel synthesis with control halogen substitution patterns in order to result in a large number of PXDDs

Synthesis of new optical sensors for determination of pH and chloride ions in reinforced concrete

Corrosion of reinforcement steel of concrete is one of the main causes of deteriorations of structures. Principally, it is an electrochemical process, which can be divided by two mechanisms – acidic and chloride initiated corrosion. Optical sensoric materials were prepared, which allow a non-destructive monitoring of these processes. The sensors are embedded into concrete structures and the signal is transported through optical fibres. pH–indicator azo dyes have been synthesised for monitoring of acidic attack. An aldehyde group has been used as dye–reactive functionality for the first time. The dyes have been covalent bound to poly(vinyl alcohol co ethylen) by acetal spacer bridge to assure long–term stability of the sensor. The colour change of such prepared pH–indicator materials occurs in the pH interval of 11 to 13 and allows to predict the acidic attack in an early stage. The response time of these sensors is in the range of seconds. The optical detection of chloride ions in alkaline media has been investigated. Benzimidazolotrimethine dyes tend to form J–aggregates under influence of chloride ion concentration in pH of 10 to 13. A suitable polymer has been developed – poly(vinyl alcohol co vinyl butyral), in which the dye is dissolved as host–guest system. Such prepared sensor material allows a precise determination of chloride concentration and shows an excellent selectivity against other anions, which are typical in the presence of concrete structures. To the best of our knowledge it is the first optical sensors for determination of chloride in alkaline media.Die Korrosion des Baustahls im Beton ist eine der Hauptursache von Schäden an Bauwerken. Es handelt sich um einen elektrochemischen Prozess, der zwei Mechanismen haben kann –säure- und chloridinduzierte Korrosion. Optische Sensoren für eine zerstörungsfreie Überwachung von Betonbauwerken sind entwickelt worden. Die Sensoren werden in Betonstrukturen eingebettet und das Signal wird über optische Fasern geführt. pH–Indikatoren auf Basis von Azofarbstoffen für die Überwachung des Säurenangriffs sind synthetisiert worden. Dabei ist zum ersten Mal eine Aldehydfunktion als reaktive Gruppe angewendet worden. Die Farbstoffe sind an poly(Vinylalkohol co Ethylen) durch eine Acetalbrücke kovalent gebunden worden, um die Langzeitstabilität des Sensors zu sichern. Die Farbänderung der pH-Indikatoren verläuft im Intervall 11 bis 13 und kann den Säurenangriff in einer frühen Phase detektieren. Die Antwortzeit dieser Sensoren beträgt einige Sekunden. Die optische Bestimmung von Chloridionen im alkalischen Milieu ist erforscht worden. Benzimidazolotrimethine Farbstoffe zeigen eine starke Tendenz zur Bildung von J-Aggregaten unter Einfluss von Chloridionen im pH-Bereich von 10 bis 13. Ein geeignetes Polymer - poly(Vinylalkohol co Vinylbutyral) - ist entwickelt worden. Der Farbstoff ist in der Polymermatrix als Host-Guest System gebunden. Dieses Sensormaterial ermöglicht eine genaue Bestimmung der Chloridionenkonzentration und zeigt eine ausgezeichnete Selektivität im Gegenwart zu anderen Anionen, die typisch sind in Betonstrukturen. Unseres Wissens nach ist das der erste optische Sensor, der die Detektion von Chloridionen im alkalischen Milieu ermöglicht

Development of optical sensor platforms based on evanescent wave interactions

A study of the principles, fabrication and behaviour of a range of optical sensor platforms based on evanescent wave interactions is presented. The platforms utilise a range of sensing techniques including absorption, fluorescence quenching and surface plasmon resonance. The absorbance-based platforms employ sol-gel-derived planar waveguides and grating couplers to launch light into guided modes, the evanescent fields of which can interrogate a sensing layer. A detailed characterisation of these integrated optic devices is presented. Two platforms employing few-mode and multimode waveguides, respectively, are described and applied to the detection of gaseous ammonia and carbon dioxide. A fluorescence-based sensor platform which utilises the anisotropy of fluorescence emitted near an interface is also presented. The platform consists of a planar waveguide coated with a sol-gel-derived fluorescent layer. A substantial amount of the analytesensitive fluorescence is captured by guided modes in the planar waveguide. This configuration is described in detail and is applied to the detection of gaseous oxygen as a proof of principle. Finally, work leading to the development of a fibre optic probe biosensor based on surface plasmon resonance is presented together with its application to the detection of red blood cells. In summary, this work highlights the feasibility of combining evanescent wave interactions and sol-gel technology to fabricate miniature sensor platforms which may be applied to the detection of a wide range of target analytes in a reliable and cost effective manner

The synthesis and characterisation of novel anionic polymethine fluorophores

Fluorescenca je pojav, ko neka snov absorbira svetlobo pri krajši valovni dolžini z višjo energijo in nato emitira svetlobo pri daljši valovni dolžini z manj energije. Snovem, ki fluorescirajo pravimo fluorofori, in kadar govorimo o organskih fluoroforih, je za le-te značilno, da imajo v svoji strukturi več konjugiranih dvojnih vezi in/ali aromatske strukture. Sintezni fluorofori imajo številne aplikacije in so vgrajeni v številne proizvode, zelo veliko pa se jih uporablja v znanosti. Zaradi razširjene uporabe pa se vseskozi razvijajo novi fluorofori. Tekom magistrske naloge je Andraž Bevk izoliral fluorofor, za katerega se je izkazalo, da sodi med cianine oz. polimetinske fluorofore, a gre za povsem nov skelet, kakor tudi novo sintezno pot. V naši magistrski nalogi smo raziskovali reakcijski mehanizem in reakcijske pogoje, pri katerih nastane opisan fluorofor. V prvem delu naloge smo sintetizirali fluorofore, strukturne analoge opisanega fluorofora, kjer smo poskušali različne reaktante in pogoje za nastanek želenega barvila. Iz poskusov sintez smo ugotovili, da je za nastanek fluorofora nujno potreben heteroaromat, ki vsebuje v svoji strukturi dušik piridinskega tipa. Kot donor metinske skupine je potreben dimetilformamid (DMF), ki služi tudi kot topilo. Da nastane želeni produkt, so potrebni kisli pogoji, ki smo jih dosegli s 4,0 M HBr v ocetni kislini (AcOH) ali 2,0 M HCl. Prav tako je za potek reakcije potrebna povišana temperatura, najbolj optimalna se je izkazala temperatura pri 60 °C, višje temperature pa niso pokazale bistveno boljšega izkoristka reakcije. V drugem delu magistrske naloge smo določali vpliv različnih reakcijskih pogojev na izkoristek reakcije s tekočinsko kromatografijo ultra visoke ločljivosti (UHPLC). Pri izvedbi analiz smo uporabili spojine 2, 4, 5 in 7. Najprej smo morali pripraviti raztopine za analizo s sistemom UHPLC, s čimer smo imeli kar nekaj težav, saj so izolirane spojine zelo netopne. Nato smo za vsako spojino razvili kromatografsko metodo. Sledile so analize, kjer smo preverjali vpliv različnih temperatur in kislin na izkoristek reakcije. Ugotovili smo, da dobimo najboljše izkoristke pri temperaturi 60 °C in s kislinama 4,0 M HBr v AcOH in 2,0 M HCl. Vsem izoliranim spojinam smo na koncu pomerili še absorpcijski in emisijski spekter, kjer smo ugotovili, da naše spojine emitirajo v območju zelene barve.Fluorescence is an optical phenomenon when the substance absorbs light at a shorter wavelength with higher energy and then emits it at a longer wavelength with less energy. Fluorophores are components that play the central role in fluorescence. The main characteristic of organic fluorophores is that in their structure there are several conjugated double bonds and/or an aromatic structure. Synthetic fluorophores have many applications in different fields, and many are also used in science. Due to their widespread use, new fluorophores are developing all the time. During the master’s thesis, Andraž Bevk isolated a new fluorophore that belongs into the cyanine / polymethine group, but with a completely new skeleton through a novel synthesis path. In our master’s thesis we researched the reaction mechanism and conditions in which the above mentioned fluorophore is formed. In the first part we synthetized fluorophores that are structural analogues of the mentioned fluorophore, where we tried different reactants and conditions for the formation of the desired dye. From our experiments it has been found that for the formation of fluorophores, a heteroaromatic ring containing a pyridine-type nitrogen is essential. As a donor of the methine group, dimethylformamide (DMF) is needed, and it is also used as a solvent. To obtain the desired product, acidic conditions are required which was achieved with 4,0 M HBr in acetic acid (AcOH) or 2,0 M HCl. Also, a higher temperature is required for the desired reaction, we found that 60 ° C is the optimum, since higher temperatures did not show a significantly better reaction efficiency. In the second part of the master\u27s thesis, the effect of different reaction conditions on the yield of the reaction was determined with ultra-high performance liquid chromatography (UHPLC). In the analysis, compounds 2, 4, 5, and 7 were used. First, we had to prepare solutions for the analysis with the UHPLC system, which proved quite difficult, since the isolated compounds were very insoluble. Then, for each compound, a chromatographic method was developed. The analysis followed, where we examined the effect of different temperatures and acids on the yield of the reaction. We found that the best yields were obtained at a temperature of 60 ° C and with acids 4,0 M HBr in AcOH and 2,0 M HCl. ?Finally, all the absorption and emission spectrums of isolated compounds were measured and we established that our compounds emit light in the green color part of the visible spectrum

Molecular Photochemistry

There have been various comprehensive and stand-alone text books on the introduction to Molecular Photochemistry which provide crystal clear concepts on fundamental issues. This book entitled "Molecular Photochemistry - Various Aspects" presents various advanced topics that inherently utilizes those core concepts/techniques to various advanced fields of photochemistry and are generally not available. The purpose of publication of this book is actually an effort to bring many such important topics clubbed together. The goal of this book is to familiarize both research scholars and post graduate students with recent advancement in various fields related to Photochemistry. The book is broadly divided in five parts: the photochemistry I) in solution, II) of metal oxides, III) in biology, IV) the computational aspects and V) applications. Each part provides unique aspect of photochemistry. These exciting chapters clearly indicate that the future of photochemistry like in any other burgeoning field is more exciting than the past

Development of Multiplexable Biosensors to Quantify the Spatiotemporal Dynamics of Rho GTPases and Protein Kinases in the Same Living Cell

Cell motility is a highly dynamic and heterogenous cellular process regulated by the coordination of multiple Rho GTPases, Src family kinases, and the mitogen-activated protein kinase (MAPK) cascades. However, it has been difficult to monitor more than two protein activities in the same cell due to the overlapping spectra of current biosensors and biological perturbations at high biosensor concentrations. Dye-based biosensors, which rely on an affinity scaffold that binds only to the activated conformation of the endogenous targets and an environment-sensing dye that changes its fluorescence properties to report the specific binder-target interactions, possess great potential to monitor multiple endogenous targets in the same cell. Here, I created novel environment-sensing dyes and exploited novel affinity scaffolds to develop multiplexable dye-based biosensors capable of quantifying the spatiotemporal dynamics of multiple Rho GTPases and protein kinases in the same cell. Src protein kinase is an upstream regulator of the Cdc42 GTPase. The coordination of Cdc42 and Src at the leading edge has not been well characterized due to lack of multiplexable biosensors to monitor Cdc42 and Src activities in the same living cell. Therefore, I developed novel near infrared merocyanine dyes and a red ratiometric merocyanine dye with an intrinsic ratiometric response that can be used to construct multiplexible biosensors. The relative timing and the subcellular localization of active Cdc42 and Src during leading edge dynamics and during pinocytosis were revealed using the new dyes. Src also plays an important role in the MAPK-mediated cell motility. However, the precise roles of MAPKKs and MAPKs at the leading edge remain poorly characterized due to the lack of sensitive biosensors for each target. By taking advantage of the specific interactions between MAPKKs and MAPKs, I developed the first substrate-based biosensor designs to report the activity of endogenous MEK1/2 and MKK3/6. I also developed a sensitive ERK1/2 biosensor based on artificial binders through collaborations with the Plűckthun group. This work will provide a foundation to study the crosstalk between Rho GTPases, Src family kinases and the MAPK cascades via multiplexed live cell imaging.Doctor of Philosoph