Desenvolvimento de um biossensor para Ácido Glutâmico

A presente dissertação tem com objetivo o desenvolvimento de um biossensor com base nos polímeros de impressão molecular para a deteção de uma molécula alvo, o ácido glutâmico que é convertido em glutamina pela glutamina sintetase, recorrendo à potenciometria. Nas células neoplásicas a glutamina não é sintetizada podendo-se considerar que o ácido glutâmico é um potencial agente anti-cancro. A técnica de impressão molécular utilizada foi a polimerização em bulk, combinando a acrilamida e a bis acrilamida com o ácido glutâmico. Para se verificar se a resposta potenciométrica obtida era de facto da molécula alvo foram preparados em paralelo com os sensores, materiais de controlo, ou seja, moléculas sem impressão molécular (NIP). Para se controlar a constituíção química dos vários sensores nomeadamente, do NIP e do polímero de impressão molecular (MIP) antes e após a remoção bem como a molécula foram realizados estudos de Espetroscopia de Infravermelhos de Transformada de Fourier (FTIR), Scanning electron microscope (SEM) e Espetroscopia de Raios X por dispersão em energia (EDS). Os materiais desenvolvidos foram aplicados em várias membranas que diferiam umas das outras, sendo seletivas ao ião. A avaliação das características gerais das membranas baseou-se na análise das curvas de calibração, conseguidas em meios com pHs diferentes, comparando os vários elétrodos. O pH 5 foi o que apresentou melhor resultado, associado a uma membrana que continha um aditivo, o p-tetra-octilphenol, e com o sensor com percentagem de 3%. Posto isto, testou-se em material biológico, urina, com as melhores características quer em termos de sensibilidade (18,32mV/década) quer em termos de linearidade (1,6x10-6 a 1,48x10-3 mol/L). Verificou-se ainda que aplicando iões interferentes na solução, estes não interferem nesta, podendo ser aplicados na amostra sem que haja alteração na resposta potenciométrica. O elétrodo é capaz de distinguir o ácido glutâmico dos restantes iões presentes na solução.

Determination of total petroleum hydrocarbons in soil from different locations using infrared spectrophotometry and gas chromatography

Total petroleum hydrocarbons (TPH) are important environmental contaminants which are toxic to human and environmental receptors. Several analytical methods have been used to quantify TPH levels in contaminated soils, specifically through infrared spectrometry (IR) and gas chromatography (GC). Despite being two of the most used techniques, some issues remain that have been inadequately studied: a) applicability of both techniques to soils contaminated with two distinct types of fuel (petrol and diesel), b) influence of the soil natural organic matter content on the results achieved by various analytical methods, and c) evaluation of the performance of both techniques in analyses of soils with different levels of contamination (presumably non-contaminated and potentially contaminated). The main objectives of this work were to answer these questions and to provide more complete information about the potentials and limitations of GC and IR techniques. The results led us to the following conclusions: a) IR analysis of soils contaminated with petrol is not suitable due to volatilisation losses, b) there is a significant influence of organic matter in IR analysis, and c) both techniques demonstrated the capacity to accurately quantify TPH in soils, irrespective of their contamination levels.

Reduced fluoresceinamine as a fluorescent sensor for nitric oxide

A new fluorescent sensor for nitric oxide (NO) is presented that is based on its reaction with a non fluorescent substance, reduced fluoresceinamine, producing the highly fluorescent fluoresceinamine. Using a portable homemade stabilized light source consisting of 450 nm LED and fiber optics to guide the light, the sensor responds linearly within seconds in the NO concentration range between about 10–750 µM with a limit of detection (LOD) of about 1 µM. The system generated precise intensity readings, with a relative standard deviation of less than 1%. The suitability of the sensor was assessed by monitoring the NO generated by either the nitrous acid decomposition reaction or from a NO-releasing compound. Using relatively high incubation times, the sensor also responds quantitatively to hydrogen peroxide and potassium superoxide, however, using transient signal measurements results in no interfering species.

Man-tailored biomimetic sensor of molecularly imprinted materials for the potentiometric measurement of oxytetracycline

A novel biomimetic sensor for the potentiometric transduction of oxytetracycline is presented. The artificial host was imprinted in methacrylic acid and/or acrylamide based polymers. Different amounts of molecularly imprinted and non-imprinted polymers were dispersed in different plasticizing solvents and entrapped in a poly(vinyl chloride) matrix. Only molecularly imprinted based sensors allowed a potentiometric transduction, suggesting the existence of host–guest interactions. These sensors exhibited a near-Nernstian response in steady state evaluations; slopes and detection limits ranged 42–63 mV/decade and 2.5–31.3 µg/mL, respectively. Sensors were independent from the pH of test solutions within 2–5. Good selectivity was observed towards glycine, ciprofloxacin, creatinine, acid nalidixic, sulfadiazine, cysteine, hydroxylamine and lactose. In flowing media, the biomimetic sensors presented good reproducibility (RSD of ±0.7%), fast response, good sensitivity (65 mV/decade), wide linear range (5.0×10−5 to 1.0×10−2 mol/L), low detection limit (19.8 µg/mL), and a stable baseline for a 5×10−3M citrate buffer (pH 2.5) carrier. The sensors were successfully applied to the analysis of drugs and urine. This work confirms the possibility of using molecularly imprinted polymers as ionophores for organic ion recognition in potentiometric transduction.

Bandwidth allocation in hexagonal wireless sensor networks for real-time communications

We present an algorithm for bandwidth allocation for delay-sensitive traffic in multi-hop wireless sensor networks. Our solution considers both periodic as well as aperiodic real-time traffic in an unified manner. We also present a distributed MAC protocol that conforms to the bandwidth allocation and thus satisfies the latency requirements of realtime traffic. Additionally, the protocol provides best-effort service to non real-time traffic. We derive the utilization bounds of our MAC protocol.

Improving quality-of-service in wireless sensor networks by mitigating “Hidden-Node Collisions”

Wireless sensor networks (WSNs) emerge as underlying infrastructures for new classes of large-scale networked embedded systems. However, WSNs system designers must fulfill the quality-of-service (QoS) requirements imposed by the applications (and users). Very harsh and dynamic physical environments and extremely limited energy/computing/memory/communication node resources are major obstacles for satisfying QoS metrics such as reliability, timeliness, and system lifetime. The limited communication range of WSN nodes, link asymmetry, and the characteristics of the physical environment lead to a major source of QoS degradation in WSNs-the ldquohidden node problem.rdquo In wireless contention-based medium access control (MAC) protocols, when two nodes that are not visible to each other transmit to a third node that is visible to the former, there will be a collision-called hidden-node or blind collision. This problem greatly impacts network throughput, energy-efficiency and message transfer delays, and the problem dramatically increases with the number of nodes. This paper proposes H-NAMe, a very simple yet extremely efficient hidden-node avoidance mechanism for WSNs. H-NAMe relies on a grouping strategy that splits each cluster of a WSN into disjoint groups of non-hidden nodes that scales to multiple clusters via a cluster grouping strategy that guarantees no interference between overlapping clusters. Importantly, H-NAMe is instantiated in IEEE 802.15.4/ZigBee, which currently are the most widespread communication technologies for WSNs, with only minor add-ons and ensuring backward compatibility with their protocols standards. H-NAMe was implemented and exhaustively tested using an experimental test-bed based on ldquooff-the-shelfrdquo technology, showing that it increases network throughput and transmission success probability up to twice the values obtained without H-NAMe. H-NAMe effectiveness was also demonstrated in a target tracking application with mobile robots - over a WSN deployment.

Quality-of-service in wireless sensor networks: state-of-the-art and future directions

Wireless sensor networks (WSNs) are one of today’s most prominent instantiations of the ubiquituous computing paradigm. In order to achieve high levels of integration, WSNs need to be conceived considering requirements beyond the mere system’s functionality. While Quality-of-Service (QoS) is traditionally associated with bit/data rate, network throughput, message delay and bit/packet error rate, we believe that this concept is too strict, in the sense that these properties alone do not reflect the overall quality-ofservice provided to the user/application. Other non-functional properties such as scalability, security or energy sustainability must also be considered in the system design. This paper identifies the most important non-functional properties that affect the overall quality of the service provided to the users, outlining their relevance, state-of-the-art and future research directions.

H-NAMe: specifying, implementing and testing a hidden-node avoidance mechanism for wireless sensor networks

The hidden-node problem has been shown to be a major source of Quality-of-Service (QoS) degradation in Wireless Sensor Networks (WSNs) due to factors such as the limited communication range of sensor nodes, link asymmetry and the characteristics of the physical environment. In wireless contention-based Medium Access Control protocols, if two nodes that are not visible to each other transmit to a third node that is visible to the formers, there will be a collision – usually called hidden-node or blind collision. This problem greatly affects network throughput, energy-efficiency and message transfer delays, which might be particularly dramatic in large-scale WSNs. This technical report tackles the hidden-node problem in WSNs and proposes HNAMe, a simple yet efficient distributed mechanism to overcome it. H-NAMe relies on a grouping strategy that splits each cluster of a WSN into disjoint groups of non-hidden nodes and then scales to multiple clusters via a cluster grouping strategy that guarantees no transmission interference between overlapping clusters. We also show that the H-NAMe mechanism can be easily applied to the IEEE 802.15.4/ZigBee protocols with only minor add-ons and ensuring backward compatibility with the standard specifications. We demonstrate the feasibility of H-NAMe via an experimental test-bed, showing that it increases network throughput and transmission success probability up to twice the values obtained without H-NAMe. We believe that the results in this technical report will be quite useful in efficiently enabling IEEE 802.15.4/ZigBee as a WSN protocol.

H-NAMe: a hidden-node avoidance mechanism for wireless sensor networks

The hidden-node problem has been shown to be a major source of Quality-of-Service (QoS) degradation in Wireless Sensor Networks (WSNs) due to factors such as the limited communication range of sensor nodes, link asymmetry and the characteristics of the physical environment. In wireless contention-based Medium Access Control protocols, if two nodes that are not visible to each other transmit to a third node that is visible to the formers, there will be a collision – usually called hidden-node or blind collision. This problem greatly affects network throughput, energy-efficiency and message transfer delays, which might be particularly dramatic in large-scale WSNs. This paper tackles the hiddennode problem in WSNs and proposes H-NAMe, a simple yet efficient distributed mechanism to overcome it. H-NAMe relies on a grouping strategy that splits each cluster of a WSN into disjoint groups of non-hidden nodes and then scales to multiple clusters via a cluster grouping strategy that guarantees no transmission interference between overlapping clusters. We also show that the H-NAMe mechanism can be easily applied to the IEEE 802.15.4/ZigBee protocols with only minor add-ons and ensuring backward compatibility with the standard specifications. We demonstrate the feasibility of H-NAMe via an experimental test-bed, showing that it increases network throughput and transmission success probability up to twice the values obtained without H-NAMe. We believe that the results in this paper will be quite useful in efficiently enabling IEEE 802.15.4/ZigBee as a WSN protocol

A comprehensive simulation study of slotted CSMA/CA for IEEE 802.15.4 wireless sensor networks

In this paper, we analyze the performance limits of the slotted CSMA/CA mechanism of IEEE 802.15.4 in the beacon-enabled mode for broadcast transmissions in WSNs. The motivation for evaluating the beacon-enabled mode is due to its flexibility for WSN applications as compared to the non-beacon enabled mode. Our analysis is based on an accurate simulation model of the slotted CSMA/CA mechanism on top of a realistic physical layer, with respect to the IEEE 802.15.4 standard specification. The performance of the slotted CSMA/CA is evaluated and analyzed for different network settings to understand the impact of the protocol attributes (superframe order, beacon order and backoff exponent) on the network performance, namely in terms of throughput (S), average delay (D) and probability of success (Ps). We introduce the concept of utility (U) as a combination of two or more metrics, to determine the best offered load range for an optimal behavior of the network. We show that the optimal network performance using slotted CSMA/CA occurs in the range of 35% to 60% with respect to an utility function proportional to the network throughput (S) divided by the average delay (D).

On the performance limits of slotted CSMA/CA in IEEE 802.15.4 for broadcast transmissions in wireless sensor networks

The IEEE 802.15.4 has been adopted as a communication protocol standard for Low-Rate Wireless Private Area Networks (LRWPANs). While it appears as a promising candidate solution for Wireless Sensor Networks (WSNs), its adequacy must be carefully evaluated. In this paper, we analyze the performance limits of the slotted CSMA/CA medium access control (MAC) mechanism in the beacon-enabled mode for broadcast transmissions in WSNs. The motivation for evaluating the beacon-enabled mode is due to its flexibility and potential for WSN applications as compared to the non-beacon enabled mode. Our analysis is based on an accurate simulation model of the slotted CSMA/CA mechanism on top of a realistic physical layer, with respect to the IEEE 802.15.4 standard specification. The performance of the slotted CSMA/CA is evaluated and analyzed for different network settings to understand the impact of the protocol attributes (superframe order, beacon order and backoff exponent), the number of nodes and the data frame size on the network performance, namely in terms of throughput (S), average delay (D) and probability of success (Ps). We also analytically evaluate the impact of the slotted CSMA/CA overheads on the saturation throughput. We introduce the concept of utility (U) as a combination of two or more metrics, to determine the best offered load range for an optimal behavior of the network. We show that the optimal network performance using slotted CSMA/CA occurs in the range of 35% to 60% with respect to an utility function proportional to the network throughput (S) divided by the average delay (D).

Sensor electroquímico seletivo para a Norfloxacina

A Norfloxacina (NFX) é um antibiótico antibacteriano indicado para combater bactérias Gram-negativas e amplamente utilizado para o tratamento de infeções no trato respiratório e urinário. Com a necessidade de realizar estudos clínicos e farmacológicos esenvolveram-se métodos de análise rápida e sensitiva para a determinação da Norfloxacina. Neste trabalho foi desenvolvido um novo sensor eletroquímico sensível e seletivo para a deteção da NFX. O sensor foi construído a partir de modificações efetuadas num elétrodo de carbono vítreo. Inicialmente o elétrodo foi modificado com a deposição de uma suspensão de nanotubos de carbono de paredes múltiplas (MWCNT) de modo a aumentar a sensibilidade de resposta analítica. De seguida um filme polímerico molecularmente impresso (MIP) foi preparado por eletrodeposição, a partir de uma solução contendo pirrol (monómero funcional) e NFX (template). Um elétrodo de controlo não impresso foi também preparado (NIP). Estudouse e caraterizou-se a resposta eletroquímica do sensor para a oxidação da NFX por voltametria de onda quadrada. Foram optimizados diversos parâmetros experimentais, tais como, condições ótimas de polimerização, condições de incubação e condições de extração. O sensor apresenta um comportamento linear entre a intensidade da corrente do pico e o logaritmo da concentração de NFX na gama entre 0,1 e 8μM. Os resultados obtidos apresentam boa precisão, com repetibilidade inferior a 6% e reprodutibilidade inferior a 9%. Foi calculado a partir da curva de calibração um limite de deteção de 0,2 μM O método desenvolvido é seletivo, rápido e de fácil manuseamento. O sensor molecularmente impresso foi aplicado com sucesso na deteção da NFX em amostras de urina real e água.

Desenvolvimento e aplicação de um biossensor para monitorização de creatinina, um biomarcador associado a doenças emergentes

Este trabalho descreve o desenvolvimento de um material sensor para creatinina por impressão molecular em estrutura polimérica (MIP) e a sua aplicação no desenvolvimento de um dispositivo de natureza potenciométrica para a determinação da molécula alvo em fluidos biológicos. A creatinina é um dos biomarcadores mais utilizados no acompanhamento da doença renal, já que é um bom indicador da taxa de filtração glomerular (TFG). Os materiais biomiméticos desenhados para interação com a creatinina foram obtidos por polimerização radicalar, recorrendo a monómeros de ácido metacríclico ou de vinilpiridina e a um agente de reticulação apropriado. De modo a aferir o efeito da impressão da creatinina na resposta dos materiais MIP à sua presença, foram também preparados e avaliados materiais de controlo, obtidos sem impressão molecular (NIP). O controlo da constituição química destes materiais, incluindo a extração da molécula impressa, foi realizado por Espectroscopia de Raman e de Infravermelho com Transformada de Fourrier. A afinidade de ligação entre estes materiais e a creatinina foi também avaliada com base em estudos cinéticos. Todos os materiais descritos foram integrados em membranas selectivas de elétrodos seletivos de ião, preparadas sem ou com aditivo iónico lipófilo, de carga negativa ou positiva. A avaliação das características gerais de funcionamento destes elétrodos, em meios de composição e pH diferentes, indicaram que as membranas com materiais impressos e aditivo aniónico eram as únicas com utilidade analítica. Os melhores resultados foram obtidos em solução tampão Piperazine-N,N′-bis(2- ethanesulfonic acid), PIPES, de pH 2,8, condição que permitiu obter uma resposta quasi-Nernstiana, a partir de 1,6×10-5 mol L-1. Estes elétrodos demonstraram ainda uma boa selectividade ao apresentaram uma resposta preferencial para a creatinina quando na presença de ureia, carnitina, glucose, ácido ascórbico, albumina, cloreto de cálcio, cloreto de potássio, cloreto de sódio e sulfato de magnésio. Os elétrodos foram ainda aplicados com sucesso na análise de amostras sintéticas de urina, quando os materiais sensores eram baseados em ácido metacrilico, e soro, quando os materiais sensores utilizados eram baseados em vinilpiridina.

Electrospun Polyaniline-Reduced Graphene Oxide Composite Nanofibers Based High Sensitive Ammonia Gas Sensor

Ammonia is an important gas in many power plants and industrial processes so its detection is of extreme importance in environmental monitoring and process control due to its high toxicity. Ammonia’s threshold limit is 25 ppm and the exposure time limit is 8 h, however exposure to 35 ppm is only secure for 10 min. In this work a brief introduction to ammonia aspects are presented, like its physical and chemical properties, the dangers in its manipulation, its ways of production and its sources. The application areas in which ammonia gas detection is important and needed are also referred: environmental gas analysis (e.g. intense farming), automotive-, chemical- and medical industries. In order to monitor ammonia gas in these different areas there are some requirements that must be attended. These requirements determine the choice of sensor and, therefore, several types of sensors with different characteristics were developed, like metal oxides, surface acoustic wave-, catalytic-, and optical sensors, indirect gas analyzers, and conducting polymers. All the sensors types are described, but more attention will be given to polyaniline (PANI), particularly to its characteristics, syntheses, chemical doping processes, deposition methods, transduction modes, and its adhesion to inorganic materials. Besides this, short descriptions of PANI nanostructures, the use of electrospinning in the formation of nanofibers/microfibers, and graphene and its characteristics are included. The created sensor is an instrument that tries to achieve a goal of the medical community in the control of the breath’s ammonia levels being an easy and non-invasive method for diagnostic of kidney malfunction and/or gastric ulcers. For that the device should be capable to detect different levels of ammonia gas concentrations. So, in the present work an ammonia gas sensor was developed using a conductive polymer composite which was immobilized on a carbon transducer surface. The experiments were targeted to ammonia measurements at ppb level. Ammonia gas measurements were carried out in the concentration range from 1 ppb to 500 ppb. A commercial substrate was used; screen-printed carbon electrodes. After adequate surface pre-treatment of the substrate, its electrodes were covered by a nanofibrous polymeric composite. The conducting polyaniline doped with sulfuric acid (H2SO4) was blended with reduced graphene oxide (RGO) obtained by wet chemical synthesis. This composite formed the basis for the formation of nanofibers by electrospinning. Nanofibers will increase the sensitivity of the sensing material. The electrospun PANI-RGO fibers were placed on the substrate and then dried at ambient temperature. Amperometric measurements were performed at different ammonia gas concentrations (1 to 500 ppb). The I-V characteristics were registered and some interfering gases were studied (NO2, ethanol, and acetone). The gas samples were prepared in a custom setup and were diluted with dry nitrogen gas. Electrospun nanofibers of PANI-RGO composite demonstrated an enhancement in NH3 gas detection when comparing with only electrospun PANI nanofibers. Was visible higher range of resistance at concentrations from 1 to 500 ppb. It was also observed that the sensor had stable, reproducible and recoverable properties. Moreover, it had better response and recovery times. The new sensing material of the developed sensor demonstrated to be a good candidate for ammonia gas determination.

Chitosan/AuNPs Modified Graphene Electrochemical Sensor for Label-Free Human Chorionic Gonadotropin Detection

A new immunosensor is presented for human chorionic gonadotropin (hCG), made by electrodepositing chitosan/gold-nanoparticles over graphene screen-printed electrode (SPE). The antibody was covalently bound to CS via its Fc-terminal. The assembly was controlled by electrochemical Impedance Spectroscopy (EIS) and followed by Fourier Transformed Infrared (FTIR). The hCG-immunosensor displayed linear response against the logarithm-hCG concentration for 0.1–25 ng/mL with limit of detection of 0.016 ng/mL. High selectivity was observed in blank urine and successful detection of hCG was also achieved in spiked samples of real urine from pregnant woman. The immunosensor showed good detection capability, simplicity of fabrication, low-cost, high sensitivity and selectivity.