Dissertations / Theses on the topic 'Chemiluminescent reactions'

The overall objectives of this thesis were to investigate the potential of the peroxyoxalate chemiluminescence (POOL) reaction for the quantitative detection of target analytes in non-aqueous matrices and to compare quantitative performance with fluorescence detection. The target analytes investigated were polycyclic aromatic hydrocarbons (PAHs) and aliphatic amines. These were selected as an important class of compounds in engine exhaust emissions and a detergent additive in diesel fuel respectively. Chapter one outlines the challenges of analysing petroleum products and engine exhaust emissions and discusses the potential of luminescence techniques, particularly chemiluminescence (CL), for the quantification of trace components. The chapter also reviews the technique of flow injection (FT) as a means of sample delivery for CL detection and as a potential technique for field deployment. Liquid chromatography techniques are described as a means of separation of complex matrices, e.g. fuels and engine exhaust particulates, in the laboratory prior to CL detection. The luminescence properties of several PAHs were investigated in Chapter Two. Optimum excitation and emission wavelengths for eleven PAHs in four different solvents were determined using a batch fluorescence technique. A FI approach was used to determine PAH concentrations using fluorescence and POCL detection. Two aryl oxalates; bis(2,4-dinitophenyl)oxalate and bis(2,4,6-trichlorophenyl)oxalate were compared for their suitability for PAH determinations and an investigation of the key variables (e.g. concentration of aryl oxalate and hydrogen peroxide, mobile phase composition and pH) affecting POCL was performed. Recommendations for the optimum conditions for the determination of PAHs by POCL detection were determined, A comparison between a photodiode based detection device and a low power (12V) photomultiplier tube was also described. In Chapter Three the procedure of using POCL detection as a post column liquid chromatography (LC) detector for PAHs has been considered. The performance of the POCL detection system was compared with wavelength programmed fluorescence. Both reversed and normal phase LC was investigated and the suitability of POCL detection with each approach was discussed. Additionally the procedure for the LC separation and analysis of SRM 1649 (Urban Dust/Organics) and SRM 1650 (Diesel Particulate Matter) was described. The relative performance of fluorescence and CL detection are discussed. Chapter four describes the principles of multivariate calibration of spectrophotometric data, and three commonly applied techniques (PCR, PLSI and PLS2). Fluorescence data was obtained for synthetic mixtures of PAHs containing two, three, four and five components. A procedure whereby individual spectra were 'glued' together before undergoing data analysis has been developed and the results obtained discussed. POCL emission spectra for five PAHs were acquired using a two-dimensional charge coupled device (CCD). The sensitivity of the CCD system toward POCL detection of PAHs and a multivariate investigation using benzo[a]pyrene and benzo[k]fluoranthene has been described. The potential of the fluorescence and CL approaches used has been discussed. Chapter five describes the aryl oxalate sulphorhodamine-101 CL reaction and its application to the determination of amines. A FI optimisation of the reaction parameters is presented together with some quantitative data for the detection of a homologous series of amines and dodecylamine (a commonly added detergent compound in diesel fuels). The application of the technique toward the detection of dodecylamine in a diesel fuel matrix and the potential as a field deployable technique was also considered.

In order to better understand the mechanism of solution manganese-based chemilumines-cence, the kinetics of a number of chemiluminescent oxidations by manganese species were studied using stopped-flow spectrophotometry. Both the kinetics of the decay of the oxi-dant and the chemiluminescence emission were followed for oxidations by permanganate, manganese dioxide sol and Mn3+(aq) of a range of organic compounds. The most detailed studies were carried out on the oxidation of the relatively simple compounds including glyoxylic acid and glyoxal under pseudo first order conditions and an acidic medium at 25oC. For permanganate under these conditions, the decay is sigmoidal consistent with autocatalysis and for managanese dioxide sol and Mn3+ pseudo first order. Simple mechanisms are suggested and compared with the experimental kinetic data. For per-manganate CL system, the following chemical kinetic model was considered: MnO4- + xsR à MnO2 (k1) MnO2 + xsR à Mn3+ (k2) MnO4- + 3Mn3+ à 4MnO2 (k3) Mn3+ + xsR à (Mn2+)*

Made available in DSpace on 2014-06-11T19:26:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2006Bitstream added on 2014-06-13T18:54:35Z : No. of bitstreams: 1 kanegae_mpp_me_arafcf.pdf: 328350 bytes, checksum: ee1587bd6ab853b184cea70a4d71dac0 (MD5)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Em hematologia, a principal aplicação da determinação da atividade da fosfatase alcalina (FA) neutrofílica é no auxílio ao diagnóstico diferencial entre leucemia mielóide crônica (LMC) e reações leucemóides neutrofílicas (RL) decorrentes de doenças mieloproliferativas, como a mielofibrose, policitemia vera ou de inflamações/ infecções. Tradicionalmente esta determinação é realizada por um ensaio citoquímico subjetivo, no qual se atribui uma pontuação (SCORE) para o nível de FA. Neste trabalho apresentamos um método quimiluminescente, objetivo, quantitativo, sensível e barato para a determinação de FA neutrofílica baseado no reagente comercial Immulite®. Leucócitos íntegros obtidos de amostras de sangue periférico de trinta e dois indivíduos saudáveis, nove portadores de LMC e nove portadores de RL foram submetidos ao protocolo otimizado. Através da determinação da emissão de luz por quatro concentrações de neutrófilos, foi possível detectar a atividade de FA por célula (inclinação - SLOPE - da curva obtida por regressão linear). Uma alta correlação foi obtida quando o método quimiluminescente (SLOPE), aqui desenvolvido, foi comparado ao citoquímico (SCORE). Obtivemos uma variação do SLOPE entre 0,61-8,49 (10-5 mV.s/célula) para amostras do grupo controle (indivíduos saudáveis), sendo que o valor da mediana foi 2,04 (10-5 mV.s/célula). Estes resultados foram estatisticamente diferentes das amostras do grupo LMC (variação: 0,07 - 1,75; mediana: 0,79) e do grupo RL (variação: 3,84 - 47,24; mediana: 9,58) (p<0,05).
In haematology the main application of the leukocyte alkaline phosphatase (LAP) assay is in distinguishing chronic myeloid leukaemia (CML) from other myeloproliferative diseases, particularly from myelofibrosis, polycythaemia or other inflammatory/infectious diseases (LR). Traditionally, this is performed by subjective cytochemical assays where a SCORE is attributed to the level of LAP. Here we present a non-subjective, quantitative, sensitive and inexpensive chemiluminescent technique for LAP determination, based on the commercial reagent Immulite®. Intact leukocytes obtained from thirty-two healthy subjects, nine CML and nine LR patients were submitted to the optimized protocol. By measuring the light emission elicited by four concentrations of neutrophils, it was possible to estimate the activity of LAP per cell (the SLOPE of the curve obtained by linear regression). A high linear correlation was found between the chemiluminescent result (SLOPE) and the cytochemical SCORE. The SLOPE for healthy individuals ranged between 0.61 and 8.49 (10-5 mV.s/cell), with a median of 2.04 (10-5 mV.s/cell). These results were statistically different from CML patients (range 0.07 - 1.75, median 0.79) and LR patients (range 3.84 - 47.24, median 9.58) (p<0.05).

Orientador: Luiz Marcos da Fonseca
Banca: Amauri Antiquera Leite
Banca: Eduardo Magalhães Rego
Resumo: Em hematologia, a principal aplicação da determinação da atividade da fosfatase alcalina (FA) neutrofílica é no auxílio ao diagnóstico diferencial entre leucemia mielóide crônica (LMC) e reações leucemóides neutrofílicas (RL) decorrentes de doenças mieloproliferativas, como a mielofibrose, policitemia vera ou de inflamações/ infecções. Tradicionalmente esta determinação é realizada por um ensaio citoquímico subjetivo, no qual se atribui uma pontuação (SCORE) para o nível de FA. Neste trabalho apresentamos um método quimiluminescente, objetivo, quantitativo, sensível e barato para a determinação de FA neutrofílica baseado no reagente comercial Immulite®. Leucócitos íntegros obtidos de amostras de sangue periférico de trinta e dois indivíduos saudáveis, nove portadores de LMC e nove portadores de RL foram submetidos ao protocolo otimizado. Através da determinação da emissão de luz por quatro concentrações de neutrófilos, foi possível detectar a atividade de FA por célula (inclinação - SLOPE - da curva obtida por regressão linear). Uma alta correlação foi obtida quando o método quimiluminescente (SLOPE), aqui desenvolvido, foi comparado ao citoquímico (SCORE). Obtivemos uma variação do SLOPE entre 0,61-8,49 (10-5 mV.s/célula) para amostras do grupo controle (indivíduos saudáveis), sendo que o valor da mediana foi 2,04 (10-5 mV.s/célula). Estes resultados foram estatisticamente diferentes das amostras do grupo LMC (variação: 0,07 - 1,75; mediana: 0,79) e do grupo RL (variação: 3,84 - 47,24; mediana: 9,58) (p<0,05).
Abstract: In haematology the main application of the leukocyte alkaline phosphatase (LAP) assay is in distinguishing chronic myeloid leukaemia (CML) from other myeloproliferative diseases, particularly from myelofibrosis, polycythaemia or other inflammatory/infectious diseases (LR). Traditionally, this is performed by subjective cytochemical assays where a SCORE is attributed to the level of LAP. Here we present a non-subjective, quantitative, sensitive and inexpensive chemiluminescent technique for LAP determination, based on the commercial reagent Immulite®. Intact leukocytes obtained from thirty-two healthy subjects, nine CML and nine LR patients were submitted to the optimized protocol. By measuring the light emission elicited by four concentrations of neutrophils, it was possible to estimate the activity of LAP per cell (the SLOPE of the curve obtained by linear regression). A high linear correlation was found between the chemiluminescent result (SLOPE) and the cytochemical SCORE. The SLOPE for healthy individuals ranged between 0.61 and 8.49 (10-5 mV.s/cell), with a median of 2.04 (10-5 mV.s/cell). These results were statistically different from CML patients (range 0.07 - 1.75, median 0.79) and LR patients (range 3.84 - 47.24, median 9.58) (p<0.05).
Mestre

Le rôle clef du fer dans le cycle biogéochimique du carbone et de l’azote dans l’océan a été mis en évidence au cours de la dernière décennie. Une des découvertes majeures récentes en océanographie biologique est la limitation de la croissance du phytoplancton par la disponibilité en fer dans au moins 40% de l’océan mondial. Or, la chimie de cet élément dans l’océan est particulièrement complexe et la forme sous laquelle il est disponible pour le phytoplancton reste encore mal connue. Plusieurs mécanismes sont utilisés par le phytoplancton marin pour améliorer la solubilité du fer en eau de mer et parvenir à absorber les quantités suffisantes en fer nécessaires à leur survie. Un de ces mécanismes implique la production de radicaux superoxyde en milieu extracellulaire, ce qui accroît la bio-disponibilité du fer en eau de mer en réduisant la forme Fe(III) sous forme Fe(II), plus bio-disponible aux cellules de phytoplancton. Les objectifs principaux de ce travail étaient de i) développer une méthode appropriée pour détecter la production de superoxyde en milieu extracellular par n’importe quelle cellule de phytoplancton marin, et ii) examiner la relation entre la production extracellulaire de superoxyde et l’absorption du fer par la cyanobactérie Trichodesmium erythraeum IMS101. Une méthode de détection du superoxyde a été développée, qui utilise du red-CLA ou du MCLA, deux sondes chimiluminescentes spécifiques à la détection du superoxyde, qui ont donné des résultats fiables, même sur de très faibles quantités d’échantillons. En effet, comparée aux autres méthodes employées, la détection de la production du superoxyde par microplaques permet de réduire le volume d’échantillon par 10, et de réduire le temps d’analyse de tréplicats d’un échantillon, d’un blanc et de trois standards à 10 minutes. De plus, cette méthode présente une large gamme de travail avec une limite de détection de 0,076 pmol/s, ce qui lui confère un grand avantage pour le travail sur le phytoplancton marin. Les taux de production de superoxyde en milieu extracellulaire par la cyanobatérie Trichodesmium erythraeum ont été mesurés en condition de laboratoire et allaient de 0,93 à 16,21 pmol/trichome/h. La limitation en fer des cellules de Trichodesmium résultat en une augmentation de ce taux de production, qui a été multiplié par un facteur 2,9 entre les cellules non limitées et les cellules limitées en fer. Il a aussi été montré que la production de superoxyde suivait un rythme diurne avec une forte augmentation du taux de production en milieu du cycle « jour», spécialement marqué pour les cultures maintenues en milieu pauvre en fer. Les taux de production extracellulaire de superoxyde et d’absorption du fer par Trichodesmium ont été mesurés simultanément sur des cultures pré-limitées ou non limitées en fer. Les taux d’absorption étaient 10 fois plus élevés pour les cultures non limitées, sauf lorsqu’un composé réducteur (acide ascorbique) était ajouté. Dans ce cas, les taux d’absorption des deux cultures étaient similaires. De plus, les deux cultures ont montré une plus grande aptitude à absorber le fer lié à des ligands faibles comme le citrate. Dans l’ensemble, les résultats ont montré une relation entre la production de superoxyde et l’absorption de fer par Trichodesmium, mais aucune influence directe entre ces deux processus n’a pu être démontrée. La méthode de détection du superoxyde par microplaque a été utilisée lors de campagnes sur la Grande Barrière de corail en Australie. L’analyse de deux blooms de Trichodesmium a montré de forts taux de production de superoxyde, en cohérence avec les analyses effectuées au laboratoire. De plus, l’utilisation de cette méthode (entre autres) a permis de démontrer une accumulation d’espèces Fe(II) en concentrations biologiquement significatives, quand la concentration en superoxyde dans l’eau de mer était inférieure à 1 nM. Par contre, lorsque cette concentration se trouvait supérieure à 1nM, la plupart des espèces réduites (Fe(II)) étaient réoxidées, ce qui résultait en un fort taux de production de peroxyde d’hydrogène du à la dismutation du superoxyde. Dans l’ensemble, cette étude a permis le développement d’une méthode de détection de la production de superoxyde par le phytoplancton marin en milieu extracellulaire qui peut être utilisée au laboratoire ou en conditions d’étude sur le terrain. Nous avons aussi démontré que les cellules de Trichodesmium erythraeum IMS101 produisent de grandes quantités de superoxyde, en particulier lorsqu’elles sont limitées en fer. L’étude des taux d’absorption du fer par ces même cellules a démontré une forte relation entre ce processus et la production de superoxyde par les cellules: ces résultats sont en accord avec l’hypothèse que le modèle d’absorption du fer par le phytoplancton marin «Fe’» serait fortement influencé par ce type d’organisme capable de modifier l’équilibre redox du milieu présent à la surface des cellules
It is hypothesised that, under iron limitation, phytoplankton cells develop biochemical mechanisms to increase their iron uptake efficiency with one of these mechanisms involving the production of superoxide in the extracellular environment that increases the bioavailability of iron in seawater by reducing Fe(III) to the more soluble Fe(II). The main objectives of this work were 1) to develop an appropriate method to detect extracellular production of superoxide by marine phytoplankton, and 2) to examine the relationship between extracellular production of superoxide and iron acquisition by Trichodesmium erythraeum. A method to measure superoxyde production is described using red-CLA and MCLA probes, yielding considerable improvement for analysis compared to other available methods. Extracellular superoxide production and iron uptake rates were measured simultaneously on iron replete and iron deplete Trichodesmium erythraeum IMS 101 laboratory cultures : iron starvation leads to a 2. 9-fold increase in superoxide production rate and 10-fold decrease in the iron uptake rate (except when a reducing compound was added) compared to iron replete cultures. Extracellular superoxide production shows a pronounced circadian rythm in iron deplete cultures, but less so in iron replete cultures. Overall, no direct impact of extracellular superoxide production by Trichodesmium is observed, but both processes are shown to be related. Both iron deplete and iron replete cultures demonstrate greater ability to uptake iron bound to weaker iron-binding ligands such as citrate. Application of the method to field studies in the Great Barrier Reef lagoon showed an accumulation of biologically significant concentrations of reduced trace metals including Fe(II) when the concentration of superoxide was lower than 1 nM. When the concentration of superoxide was higher than 1 nM, most of the reduced species were oxidised resulting in high rates of hudrogen peroxide production rates, consistent with laboratory studies. Overall, this thesis permitted the development of a method to detect superoxide production rates by marine phytoplankton cells that could be used routinely in field studies. The observations are in accord with the conclusion that fit the ongoing hypothesis that the extablished Fe' uptake model for phytoplankton would be strongly influenced by such organisms that are able to modify the redox equilibrium of the solution at their cells surface

Since the late 1990s our group has been working with groups in chemistry department at the University of Texas at Austin on a project referred as "Electronic Taste Chip," a MicroElectroMechanical System (MEMS) based miniaturized microfluidic chemical sensor with multianalyte detection capabilities. By integrating optical detection mechanism directly onto the silicon chip a cost effective, compact, and portable sensor can be realized enabling use of these chips out of conventional laboratory environment. Addition to the integration a noble approach of accessing a photodiode with non-contact powerless RFID type readout is presented. By doing so a packaged photodiode can be interrogated without direct electrical contact, enhancing the portability even further for a sensor operated in aqueous medium. First background information regarding the project as well as design and integration criteria is presented followed by demonstration of non-contact RFID-type readout of a photodiode. Detailed discussion on the development of process integration scheme is discussed along with the measurements verifying the performance of the fabricated photodiode. During this investigation normally overlooked design criteria of collection efficiency, the effect of how a target element is to be delivered to a detection mechanism on the overall performance of the sensor, is addressed and discussed.

碩士
國立交通大學
應用化學系碩博士班
104
Constructing robust biochemical circuits can help researchers to understand fundamental mechanisms of biocatalytic co-operativity, and to develop new applications of biocatalysis. Replicating chains of biochemical reactions in vitro or redesigning biochemical networks – using biomolecular building blocks – is a new avenue of exciting fundamental and applied research. Enzymes are omnipresent. They speed up numerous reactions in the living organisms. For example, they are involved in biosynthetic pathways, energy metabolism, and circadian clocks. In the present work, we have developed enzymatic amplification circuits, which could be used in synthetic biology and bioengineering. We have implemented mass spectrometry (MS) as well as a simple low-cost chemiluminescence detection system integrating a digital reflex camera and a microtiter plate to study these circuits. In the first part of the project, we developed a chemical energy buffer system using two different enzymes (pyruvate kinase and adenylate kinase). Biocatalytic reactions often require the supply of chemical energy and phosphate groups in the form of adenosine triphosphate (ATP). In fact, supplying chemical energy to cell-free biosynthetic systems is one of the biggest challenges of synthetic biology. Shortage of energy-rich substrates (such as ATP) leads to low reaction yields. Therefore, a continuous supply of energy-rich substrates to biosynthetic reactions, carried out in vitro, has to be secured. By employing the real-time MS approach, conditions of multi-enzyme reactions could be optimized ensuring high yields of the target biosynthetic processes. In the second part of the project, we developed a biochemical timer reaction. The timer is triggered by small amounts of ATP or adenosine diphosphate (ADP). The nucleotides are spontaneously amplified. When the concentration of ATP reaches a certain level, light is emitted. The time from the start of the reaction to the appearance of light is related to the initial concentration of adenosine nucleotides (e.g. ATP or ADP). Validity of the observed dependence of the luminescence raise time on the concentration of the trigger nucleotide has been verified with kinetic models. Using the simple and low-cost apparatus, precise measurements could be carried out at optimal conditions. Because this method relies on time measurement (not light intensity measurement), it does not require calibration of the optical detector. Due to the emission of light, the effect of the reaction can easily be observed by eye. The proposed timer reaction concept can be used in the bioengineered systems, in which time of response needs to be linked with the concentration of a biomolecule. Although our work has fundamental aspects, its outcomes can also have implications on the applied science (biosynthesis, biosensing). The amplification circuits can be used to stabilize levels of energy-rich nucleotides in biosynthetic protocols, or to determine their concentrations based on time-of-luminescence.

The use of chemiluminescence measurements to monitor a range of combustion processes has been a popular area of study due to their reliable and cost-effective nature. Electronically excited carbon dioxide (CO2*) is known for its broadband emission, and its detection can lead to valuable information; however, due to its broadband characteristics, CO2* is difficult to isolate experimentally, and the chemical kinetics of this species is not well known. Although numerous works have monitored CO2* chemiluminescence, a full kinetic scheme for the species has yet to be developed. A series of shock-tube experiments was performed in H2-N2O-CO mixtures highly diluted in argon at conditions where emission from CO2* could be isolated and monitored. These results were used to evaluate the kinetics of CO2*, in particular, the main CO2* formation reaction, CO + O + M CO2* + M (R1). Based on collision theory, the quenching chemistry of CO2* was determined for eleven common collision partners. The final mechanism developed for CO2* consisted of 14 reactions and 13 species. The rate for R1 was determined based on low-pressure experiments performed in two different H2-N2O-CO-Ar mixtures. Final mechanism predictions were compared with the experimental results at low and high pressures, with good agreement seen at both conditions. Peak CO2* trends with temperature as well as overall CO2* species time histories were both monitored. Comparisons were also made with previous experiments in methane-oxygen mixtures, where there was slight over-prediction of CO2* experimental trends by the mechanism.Experimental results and mechanism predictions were also compared with past literature rates for CO2*, with good agreement for peak CO2* trends, and slight discrepancies in overall CO2* species time histories. Overall, the ability of the CO2* mechanism developed in this work to reproduce a range of experimental trends represents an improvement over existing models.