Submitted by Aelson Maciera (aelsoncm@terra.com.br) on 2017-05-11T17:29:15Z No. of bitstreams: 1 TeseTAN.pdf: 1864712 bytes, checksum: 75782f61cc6526aa709949101798707e (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2017-05-18T20:22:16Z (GMT) No. of bitstreams: 1 TeseTAN.pdf: 1864712 bytes, checksum: 75782f61cc6526aa709949101798707e (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2017-05-18T20:22:25Z (GMT) No. of bitstreams: 1 TeseTAN.pdf: 1864712 bytes, checksum: 75782f61cc6526aa709949101798707e (MD5) / Made available in DSpace on 2017-05-22T20:17:57Z (GMT). No. of bitstreams: 1 TeseTAN.pdf: 1864712 bytes, checksum: 75782f61cc6526aa709949101798707e (MD5) Previous issue date: 2016-06-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Bovine babesiosis and anaplasmosis are distinct diseases, which constitute the syndrome called tick fever, characterized by infection of red blood cells. Its distribution in herds causes the reduction in productivity and high economic losses to livestock worldwide. Anaplasma marginale, Babesia bovis, and Babesia bigemina are the predominant species in Brazil. Rhipicephalus (Boophilus) microplus is the biological vector of babesiosis and anaplasmosis may be transmitted by ticks, hematophagous flies, and fomites. It is known that the water buffalo can become infected with Babesia and Anaplasma, but little is known the extent of the infection and how it affects the health of these animals. Thus, the present study aimed to determine the frequency and the level of infection by B. bovis, B. bigemina and A. marginale in 108 water buffalo (50 calves and 58 adult females), naturally infested with the tick R. (B). microplus raised in farms located in areas of endemic stability in São Paulo state. From each animal, a blood sample from the jugular vein was taken for DNA extraction and packed cell volume (PCV) determination. Samples from auricular vessels were taken for blood smears. The body temperature was measured with a mercury thermometer column. Electrochemical impedance spectroscopy technique was used to differentiate the serum of uninfected animals from the serum of animals infected with B. bovis. To this end, antigens derived from culture were immobilized on gold electrodes (150 nm thick) to give a biosensor device using the compound [Fe (CN) 6] 3- / 4-proof redox. The peripheral blood smears were stained with May-Grunwald-Giemsa for research of hemoparasites by optical microscopy. DNA extractions were performed using the Easy DNA TM Kit (Invitrogen). DNA amplification protocols were tested with primers specific to B. bigemina, B. bovis, and A. marginale using nested PCR (nPCR) and quantitative real-time PCR (qPCR). qPCR was used to estimate the number of copies of the gene cytochrome b (mt-cytB) of both babesias and Anaplasma msp 1b gene in all samples. Merozoites of B. bigemina were seen in blood smears of three calves from the Alambari herd (all less than 0.1 of parasitemia). Molecular techniques, nPCR, and qPCR, were more sensitive in the detection of parasites that direct examination of the blood smears and the frequencies of infection were 20:37% and 100% for B. bovis and 59.26% and 100% to B. bigemina, respectively. CN of the mt-cytB gene of B. bovis and B. bigemina showed significant effects (p <0.05) of herd age, species, and their interaction. The CN values were higher (p≤0.05) for B. bovis (2.81 ± 0:07) when compared to B. bigemina (2.61 ± 12:07) and A. marginale (0:57 ± 0:07). These data suggest a high frequency of infection by B. bovis and B. bigemina in the population of water buffalo studied. Preliminary testing of diagnosing infection with B. bovis device showed changes in the impedance in the system used and clearly demonstrated that the biosensor can detect infected animals, which can be exploited for rapid detection of B. bovis infections and also extended for the test to other parasites. / A babesiose e anaplasmose bovina são enfermidades distintas, que constituem o complexo chamado de Tristeza Parasitária Bovina (TPB), caracterizada por infecção das células vermelhas do sangue. Sua distribuição nos rebanhos ocasiona redução na produtividade e grandes perdas econômicas para a pecuária mundial. Anaplasma marginale, Babesia bovis e Babesia bigemina são as espécies prevalentes no Brasil. O carrapato Rhipicephalus (Boophilus) microplus é o vetor biológico das babesioses, e a anaplasmose, além do carrapato pode ser transmitida por moscas hematófogas ou fômites. Sabe-se que, os búfalos podem se infectar com os agentes da TPB, porém pouco é conhecido a extensão dessa infecção e como ela afeta a saúde desses animais. Assim o presente estudo teve o objetivo de determinar a frequência e o nível de infecção por B. bovis, B. bigemina e A. marginale em 108 búfalos d’água (50 bezerros e 58 fêmeas adultas), naturalmente infestados com o carrapato R. (B). microplus, oriundos de fazendas localizadas em áreas endêmicas para as babesioses no estado de São Paulo. Foram colhidas amostras de sangue dos capilares auriculares para confeccção de esfregaços em lâminas de vidro e sangue da veia caudal para extração de DNA e determinação do volume globular (VG) pela técnica do microhematócrito. A técnica de espectroscopia de impedância eletroquímica foi realizada com o soro dos bovinos, com o objetivo de diferenciar animais saudáveis dos animais infectados com B. bovis. Para tanto, antígenos provenientes de cultura foram imobilizados em eletrodos de ouro (150 nm de espessura) dando origem a um dispositivo biossensor utilizando o composto [Fe (CN)6]3-/4-como prova redox. Os esfregaços de sangue periférico foram corados com May Grunwald-Giemsa para a pesquisa dos hemoparasitas por meio de microscopia óptica. As extrações de DNA foram feitas usando o Easy-DNATM Kit (Invitrogen). Foram testados protocolos de amplificação do DNA com primers específicos para B. bigemina, B. bovis e A. marginale por meio de Nested PCR (nPCR) e PCR em tempo real quantitativo (qPCR), usado para estimar o número de cópias (NC) do gene do citocromo b (mt-cytB) de ambas as babesias e do gene de superfície 1b (msp1b) de Anaplasma em todas as amostras. Foram visualizados merozoítas de B. bigemina nos esfregaços sanguíneos de três bezerros do rebanho de Alambari (todos com menos de 0,1 de parasitemia). As técnicas moleculares, nPCR e qPCR foram mais sensíveis para a detecção dos parasitas que o exame direto das lâminas, sendo que as frequências de infecção foram de 20.37% e 100% para B. bovis e 59.26% e 100% para B. bigemina, respectivamente. O NC do gene do mt-cytB de B. bovis e B. bigemina mostrou efeitos significativos (p<0.05) para rebanho-idade, espécies e sua interação. Os valores de NC foram superiores (p≤0.05) para B. bovis (2.81 ± 0.07) quando comparado a B. bigemina (2.61 ± 0.07) e A. marginale (0.57 ± 0.07). Estes dados sugerem uma elevada frequência de infecção por B. bovis e B. bigemina na população de búfalos estudada. Os testes preliminares do dispositivo de diagnóstico da infecção por B. bovis mostraram alterações na impedância do sistema usado e evidenciaram claramente que o biossensor é capaz de diferencia/detectar os animais infectados, podendo ser explorado para a detecção rápida da B. bovis e estendido também para a detecção de outros parasitas.

Submitted by Ana Guimarães Pereira (agpereir@ucs.br) on 2017-02-24T13:45:52Z No. of bitstreams: 1 Dissertacao Caroline Menti.pdf: 287733 bytes, checksum: 1e558da754c143ffdcf1b15be9ec54fc (MD5) / Made available in DSpace on 2017-02-24T13:45:53Z (GMT). No. of bitstreams: 1 Dissertacao Caroline Menti.pdf: 287733 bytes, checksum: 1e558da754c143ffdcf1b15be9ec54fc (MD5) Previous issue date: 2017-02-24 / Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul, FAPERGS.

Made available in DSpace on 2014-06-11T19:29:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-04-25Bitstream added on 2014-06-13T20:38:25Z : No. of bitstreams: 1 prado_tm_me_araiq.pdf: 1251787 bytes, checksum: e701c01c70d3c55f076e7d02386ea0b5 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O presente trabalho mostra o desenvolvimento do primeiro biossensor descrito na literatura para determinação de antibióticos β-lactâmicos, usando pasta de carbono modificada com a enzima β-lactamase, e sua aplicação em amostras de leite de vaca in natura. A partir de estudos prévios de otimização da preparação do biossensor, o grafite em pó foi ativado com carbodiimida para permitir a ligação covalente da enzima β-lactamase que, junto com o mediador de elétrons ftalocianina de cobalto ofereceram variações na corrente catódica na presença da penicilina G, sendo esta escolhida para representar os antibióticos β-lactâmicos. Utilizando este procedimento para a construção dos biossensores, foi possível registrar voltamogramas cíclicos e amperogramas que permitiram quantificar a benzilpenicilina (penicilina G) em condições de análise também otimizadas, que incluíram estudo da quantidade de mediador na pasta; o pH, tipo e concentração do eletrólito usado para realização das medidas e o potencial aplicado na amperometria. Com o método otimizado foi possível detectar penicilina G em amostras de leite in natura, fortificadas com o antibiótico, com alta exatidão (erro relativo de 1%) e boa precisão (desvio padrão relativo de 8,3%, n = 3), mostrando que o biossensor desenvolvido é uma ferramenta promissora para detecção de penicilinas, e que ao serem realizados mais estudos para diminuir seu limite de quantificação, poderá se tornar uma um método de análise alternativo aos kits comerciais existentes / This work describes the development of the first biosensor described in the literature for the determination of β-lactam antibiotics using a carbon paste electrode modified with the enzyme β-lactamase, and its application in samples of fresh cow's milk. After optimizing the biosensor preparation procedure, the graphite powder was activated with carbodiimide to allow covalent binding of the enzyme β-lactamase, which together with the electron mediator, cobalt phthalocyanine, offered variations in cathodic currents in the presence of penicillin G, which was chosen as representative of the β-lactam antibiotics. Using this procedure for the construction of biosensors, it was possible to record cyclic voltammograms and amperograms that enabled quantification of benzylpenicillin (penicillin G) under analytical conditions that had been optimized in terms of the amount of mediator in the paste, the pH, the type and concentration of the electrolyte used in the measurements, and the applied amperometric potential. With the optimized method, it was possible to detect penicillin G in samples of fresh milk fortified with the antibiotic, with high accuracy (error of 1%) and adequate precision (RSD of 8.3%, n=3), demonstrating that the proposed biosensor is a promising tool for the detection of penicillins. Once quantitation limits have been further improved, the analytical method could become an alternative to existing commercial kits

Made available in DSpace on 2014-06-11T19:29:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-08-18Bitstream added on 2014-06-13T19:38:08Z : No. of bitstreams: 1 boni_tc_me_araiq.pdf: 641126 bytes, checksum: 96c81cb1d84e4b33d5cd556438ad7458 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Este trabalho descreve o desenvolvimento e aplicação de um sensor biomimético descartável com transdução amperométrica, sensível a hidroquinona (HQ). Para isto, eletrodos impressos de grafite – SPE (FS-1, Florence sensors®) foram modificados com um catalisador biomimético do sítio ativo da tirosinase, uma cupro-enzima. O complexo de cobre usado foi o cloreto de tris-2,2’-bipiridil cobre II, [Cu(dipy)3]Cl2. Para efeitos de comparação também foram preparados sensores empregando eletrodos de carbono vítreo convencionais (GC). Os sensores foram preparados modificando os eletrodos SPE e GC com membrana de Nafion® dopada com o complexo de cobre. Para tal, foram misturados em aparelho de ultrassom, 100 μL de uma solução de complexo 5 mg mL-1, preparada em dimetilformamida (DMF) com 50 μL de solução de Nafion® a 5 % (v/v). A seguir, 100 μL da mistura foram colocados na superfície do GC e 10 μL sobre o SPE. A resposta do sensor à base de SPE (sensor- SPE) foi otimizada usando amperometria. Obtendo-se as melhores respostas em tampão Pipes 0,01 mol L-1 contendo 60 μmol L-1 de H2O2 e aplicando potencial de -300 mV vs Ag/AgCl. Sob estas condições o sensor-SPE mostrou uma faixa de resposta entre 6,0 x 10-5 e 5,6 x 10-4 mol L-1, sensibilidade de 5593,6 μA L mol-1, limite de detecção e quantificação de 5,4 x 10-6 e 1,80 x 10-5 mol L-1, respectivamente. Tratando-se de um sensor biomimético seu comportamento hiperbólico foi confirmado, e a constante aparente de Michaellis-Menten foi calculada através do gráfico de duplo recíproco obtendose um valor de 5,6 x 10-5 mol L-1, indicando uma alta afinidade do complexo pela HQ. Estudos voltamétricos também foram realizados visando à caracterização eletroquímica do sistema proposto. A seletividade do sensor foi estudada em vários compostos fenólicos... / This paper describes the development and application of a biomimetic sensor disposable with amperometric transduction, sensitive to hydroquinone (HQ). For this, printed graphite electrodes - SPE (FS-1, Florence sensors®) were modified with a biomimetic catalyst of the active site of tyrosinase, a cupro-enzyme. The copper complex used was the tris–2,2'-bipyridil cooper (II) chloride, [Cu(dipy)3]Cl2. For comparison were also prepared sensors employing conventional glassy carbon electrodes (GCE). The sensors were prepared by modifying the SPE and GCE electrodes with Nafion® membrane doped with the copper complex. For this, were mixed in an ultrasonic apparatus, 100 μL of 5 mg mL-1 complex solution, prepared in dimethylformamide (DMF), with 50 μL of Nafion® 5% (v/v) solution. Then 100 μL of this mixture were placed on the surface of GCE and 10 μL on the SPE. The response of the SPE-based sensor (sensor-SPE) was optimized using amperometry. Obtaining the best responses in 0.01 mol L-1 Pipes buffer containing 60 μmol L-1 H2O2 and applying potential of -300 mV vs Ag/AgCl. Under these conditions the SPE-sensor showed a response range between 6.0 x 10-5 and 5.6 x 10-4 mol L-1, sensitivity of 5593.6 μA L mol-1, limit of detection and quantification of 5.4 x 10-6 and 1.80 x 10-5 mol L-1, respectively. In this case, the hyperbolic behavior of the sensor response was confirmed, and the Michaelis-Menten apparent constant was calculated by the Lineweaver-Burk graph, obtained a value of 5.6 x 10-5 mol L-1, indicating a high affinity of the complex for the HQ. Voltammetric studies were also conducted to the electrochemical characterization of the proposed system. The selectivity of the sensor was studied in some phenolic compounds and the reproducibility in the sensors construction was evaluated as the relative standard deviation... (Complete abstract click electronic access below)

A caracterização das propriedades ópticas de moléculas de DNA, tais como a absorção e a fluorescência por excitação, são importantes para a determinação de parâmetros usados no desenvolvimento de biossensores fotônicos. O estudo da absorção óptica do DNA, obtido por diferentes métodos tem se mostrado muito eficiente na determinação do grau de pureza do material genético obtido por amplificação, ou extração e purificação do DNA total. Por outro lado, a fluorescência por excitação a partir de um marcador cromóforo é uma técnica importante em processos de quantificação de massa, em técnicas tais como a eletroforese em gel de agarose. Devido à baixa fluorescência de moléculas de DNA na região visível do espectro, entre 500-600nm, utiliza-se destes marcadores que se ligam à molécula e que são opticamente ativos nesta região para detecção de sua emissão de fluorescência. Neste trabalho foi realizado um estudo da fluorescência do DNA, obtido a partir de uma amplificação por transcriptase reversa do RNA (RT-PCR), na região de 400nm a 600nm, sem adição de marcador e utilizando excitação por um e dois fótons (405nm e 800nm) através da técnica de microscopia confocal. As amostras contendo solução de dsDNA (237ng/&mu;L) foram depositadas sobre um filme de prata de 200nm de espessura que também é crescido previamente sobre um substrato de vidro. Sobre o filme metálico é fabricado nanoestruturas metálicas por de litografia por feixe de íons com um microscópio de duplo feixe FEI Quanta 3D 200i. As nanoestruturas são formadas por arranjos concêntricos de anéis com diâmetros de até 20 &mu;m, largura 50nm e separados por 400nm. Quando a excitação do material genético ocorre sobre a nanoestrtutura o laser gera na nanoestrutura plasmon-polaritons de superfície (SPP) que interagem com as moléculas de dsDNA na solução. Observa-se que nas regiões onde as nanoestruturas são fabricadas que a intensidade de fluorescência da macromolécula é muito maior do que a obtida fora da estrutura e sobre o filme metálico. Os efeitos da interação entre SPPs e as moléculas aumentam a atividade óptica (taxa de emissão) e podem servir como base para a fabricação de sensores fotônicos ultrasensíveis. Concluindo, os efeitos dos campos plasmônicos sobre o fluoróforo são significativos e foram observados pela diminuição do tempo de vida das moléculas e o aumento da sua fluorescência. / The characterization of the optical properties of DNA molecules, such as absorption and excitation fluorescence, is important to the determination of the parameters used for the preparation of photonic biosensors. The study of optical absorption of DNA, obtained through different methods, currently has a high sensitivity to determine the degree of purity of the genetic material obtained by amplification, extraction and purification of the total DNA. On the other hand, excitation fluorescence using a marker is an important technique in mass quantification processes together with techniques such as agarose gel electrophoresis. Because of low fluorescence of DNA molecules, in the visible region of the spectrum, between 500-600nm, the use of labels that bind to the molecule are critically for the detection of their fluorescence emission. In this work we studied the DNA fluorescence, obtained from a RNA reverse transcriptase (RT-PCR) amplification, in the region from 400nm to 600nm, without the addition of a marker as a fluorophore and using confocal microscopy with one and two photons (405nm and 800nm). The solutions of dsDNA (237ng/&mu;L) were dropped on a silver film with 200nm tackiness deposited on a glass substrate. In the silver film nanostructures were fabricated ion beam lithography with FEI Quanta 3D 200i dual beam microscope. The nanostructures are formed by concentric arrangements of rings with diameters up to 20 &mu;m, width 50nm and separated by 400nm. When the excitation of the genetic material occurs on a nanostructure an excited surface plasmon-polaritons (SPP) is responsible for the DNA excitation. It is observed in these regions an increase of the fluorescence intensity many times higher than one obtained out of the nanostructure on the silver film. The effects of the interaction between SPPs and molecules increase the optical activity of the molecule (emission rate) and can serve as the basis of photonic sensors. Concluding, the effects of the plasmon fields on the fluorophore are significant and were observed by decreasing the life time of the molecules and the increasing of their fluorescence.

Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul, FAPERGS.

O desenvolvimento de sensores eletroquímicos para glicose integrados a cateteres tem como requisitos básicos a sua miniaturização e funcionamento por períodos relativamente longos no ambiente intravenoso. A função de tais sensores é o acompanhamento de forma continuada e em tempo real, a evolução clínica de pacientes internados em unidades de terapia intensiva (UTIs). Dentre os sensores para glicose, os biossensores amperométricos baseados na reação enzimática de glicose oxidase se mostrou o mais promissor. Diferentes procedimentos para a construção de tais microssensores implantáveis foram desenvolvidos. Um conjunto de eletrodos, constituído fios de platina (mais adiante cobre, com diâmetro 0,18 mm) e de prata (diâmetro 0,20 mm) revestidos com poli-vinil-formol foram posicionados no interior de uma agulha de aço inoxidável (30,0 mm de comprimento e 0,80 a 1,2 mm de diâmetro) e fixados com resina epóxi, constituindo um dispositivo único com os eletrodos de trabalho, referência e auxiliar, respectivamente. A otimização do sensor envolveu a platinização eletroquímica da superfície do eletrodo de trabalho, o que aumentou a sua área efetiva e favoreceu a deposição do material enzimático. A presença de um detergente não iônico favoreceu a formação de um filme uniforme de enzimas e a eletropolimerização de 1,2-diaminobenzeno (em presença de albumina de soro bovino) foi utilizada para a formação de um filme com a característica de minimizar a interferência de espécies neutras. A adição de um filme de Náfion® à superfície do sensor aumentou a seletividade. O sensor resultante caracterizou-se por possuir um tempo de resposta curto (~6 s), linearidade de 1,0 até 12,5 mmol dm-3 com um limite de detecção de 1,0 mmol dm-3 , diminuição na resposta de espécies eletroativas (ácido ascórbico 2,0% e paracetamol 10,5% em relação à glicose) e uma vida útil superior a sete dias, em tampão fosfato 0,05 mol dm-3 . Para possibilitar o implante de tais sensores, foram exploradas diferentes metodologias de esterilização, sendo a mais favorável a irradiação com acelerador de elétrons com doses acumulativas. Sensores revestidos com acetato de celulose e Náfion® (a melhor condição) apresentaram perda de atividade da ordem de 15%, após serem irradiados. / The development of electrochemical sensors for glucose integrated into catheters has as basic requisite its miniaturization and the requirement of functioning for relatively long periods in the intravenous environment. The function of such sensors is the continuous monitoring on real time of the clinical evolution of patients hospitalized in intensive therapy units (UTIs). Amongst the sensors for glucose, amperometric biosensors based on the enzymatic reaction of glucose oxidase has been considered as the most promising ones. Different procedures for the construction of such microsensors to be implanted have been developed. A set of electrodes, constituted by a platinum wire (along this work, it was substituted by copper wires) with 0.18 mm diameter and a silver wire with 0.20 mm diameter coated with poly-vinyl-formol were positioned inside a stainless needle (30.0 mm length and 0.80 to 1.2 mm diameter) and fixed with epoxy resin, resulting in a single device containing the working, reference and auxiliary electrodes, respectively. The optimization of the sensor involved the electrochemical platinization of the working electrode surface, increasing its effective area and favoring the deposition of the enzymatic material. The presence of a non-ionic detergent favored the formation of a uniform film of enzyme and the electropolymerization of 1,2-diaminobenzene (in the presence of albumin and bovine serum) was used for the formation of a film able to ® interference of neutral species. The addition of a Nafion® film to the sensor surface increased its selectivity. The resultant sensor was characterized for its short time -3 response (~6 s), linearity between 1.0 and 12.5 mmol dm-3 with a detection limit of 1.0 mmol dm-3 , reduction in the interference of electroactive species (2.0% ascorbic acid and 10.5% paracetamol in relation to glucose) and a useful life larger than seven days, in 0.05 mol dm-3 phosphate buffer. To make possible the implantation of such sensors, different methodologies of sterilization were explored, being the most favorable the irradiation with electron accelerator using accumulating doses. Sensors coated with cellulose acetate and Nafion® (the best condition) presented loss of activity (around 15%), after the irradiation.

Submitted in fulfillment of the requirements of the Degree in Chemistry, Durban University of Technology, 2017. / High prevalence and mortality cases of prostate cancer (PCa) have increased around the world, particularly in developing countries. Several forthcoming factors have been revealed nowadays, one of them is due to the incapability of the diagnostic methods to produce reliable results, which impacts negatively on cancer-treatment. However, a sensitive diagnosis of PCa cells remains a challenge in the field of biosensors. Emerging whole-cell detection as biosensing targets has opened up avenues for successful cancer diagnostics, due to high selectivity among other cells. A switchable and flexible surface-based graphene material is one of the techniques that revolutionized smart biodevice platforms in biosensor technology. In this present study, a covalently linked poly-(N-isopropylacrylamide) (PNIPAM) to graphene oxide surface has been employed as “on/off”-switchable aptamer-based sensor for the detection of PC3 whole-cancer cell. The constructed surface has benefitted from PNIPAM, as the thermal-stimulus agent, which allows the coil-to-globule transitions by triggering temperature changes. When the system is above its lower critical solution temperature (LCST) of 32oC, PNIPAM will exist as hydrophobic -globular state providing an “on” binding region for the whole-cell, reaching the interactions on the biosurface. The “off” binding systems is only possibly when the PNIPAM turns into extended-state by lowering its temperature below LCST. The first principle studies have successfully characterized the electronic behavior with particular emphasis of PNIPAM monomer functions along with the description of the structural energetics of complex through density functional theory (DFT). Docking studies have further been performed to predict a plausible binding aptamer toward the protein-representative PCa cell. To better understand the prospect of an aptamer-based tunable biosensor, molecular dynamics (MD) highlighted the behavior of PNIPAM-grafted GO in exhibiting a globular and extended conformations at above and below LCST, permitting the biomolecules to interact with each other as well as to avoid interactions, respectively. Experimental studies have been included to validate the theoretical predictions by fabricating real-biosensor systems using electrochemical impedance technique, resulting a low-detection limit down to 14 cells/mL. Engagement between theoretical and experimental studies delivered an enhanced tunable-biosensor performance for the detection of whole cell prostate cancer. / M

D.Phil. (Chemistry) / The problem of maintaining good quality of water for domestic use and for aquatic life remains a challenge. Water sources are often contaminated with pollutants from natural sources such as volcanic eruptions and by human activities such as manufacturing industries, mining, water-purification processes, agricultural activities and a vast number of other activities. Water-purification processes used by municipal authorities are designed to remove most of the pollutants but some trace amounts will always remain and have been detected in drinking water and treated waste water reservoirs. These trace amounts pose a threat to human health and the well-being of aquatic life. The detection of these trace amounts of pollutants is often carried out by laboratory-based techniques that require sophisticated, expensive instruments and often require extensive sample preparation and pre-concentration. Simple, quick and in-field detection methods are necessary especially for remote small communities with limited or no access to laboratories. Optical detection systems offer hope as a solution to this problem. In this work newly developed fluorescence-based molecular sensors for the detection of pollutants in water were developed, characterised and tested for their sensing abilities towards organic and inorganic pollutants. The fluorescent probes for organic pollutants were designed based on the host-guest chemistry of the cyclodextrin molecule. Azo dye-modified β-cyclodextrins were synthesised and linked via ethylene glycol and epichlorohydrin to produce the sensors that were then tested for their sensing response towards chlorophenols and small aliphatic chlorinated alkanes which are often formed during the disinfection of water in the purification process. The sensor molecules were characterised by UV-Vis, FT-IR and 1D and 2D NMR spectroscopy. The amount of cyclodextrin in each sensor molecule was quantified using the anthrone method (67%) as well as by 1H-NMR spectroscopy (72%). To demonstrate the host-guest interaction of the sensor molecules, isothermal titration calorimetry (ITC) was used. ITC measurements showed that modifying β-cyclodextrin and using linkers did not alter its host-guest interaction with guest molecules as demonstrated by the stoichiometry, n, stability (or binding or association) constant (K) and thermodynamic parameters of the interaction. The sensor molecule linked via ethylene glycol showed selectivity towards 4- chlorophenol among the chlorophenols investigated and has the potential to be used in a sensor for the detection of 4-chlorophenol. The sensor molecule linked via epichlorohydrin showed sensitivity towards chloroform, a typical disinfection by-product. These experimental results showed that the sensor molecules could be used for quick on-field detection of chlorinated organic compounds in water. Sensor molecules for inorganic pollutants were based on the complex formation of crown ethers with metal ions. The sensor was formed by modifying a dibenzo-18- crown-6 ether molecule with an azo dye. The sensor was then characterised using UV-Vis spectrophotmetry, FT-IR and NMR spectroscopies as well as mass spectrometry and CHNS elemental analysis. The sensor molecule was then subjected to different metal ions and the fluorescence change of the probe observed. Interestingly, the sensor was highly sensitive and selective to mercury (II) and Cu (II) ions in water. Mercury (II) is one of the most hazardous heavy metals among the heavy-metal ions found in environmental waters and its early detection in water sources is important. The synthesised molecular sensor can therefore be incorporated into a simple hand-held gadget with a light source and be used for on-field detection of mercury (II) ions in remote areas.

No description available.