Pré-concentração de cádmio com Saccharomyces cerevisiae e determinação em águas fluviais usando espectrometria de emissão óptica com plasma indutivamente acoplado

A preconcentration method based on the use of Saccharomyces cerevisiae as sorbent material is proposed for the determination of Cd(II) in river water. The solid phase extraction was performed in batch mode and the determination of the analyte in the solid phase was easily carried out by introducing a slurry of the yeast (0.0625 g / 2.5 mL) directly into the ICP OES. A limit of detection of 0.11 µg L-1 and a sample throughput in the range of 4 - 54 sample h-1 were obtained. Determinations of cadmium in a certified sample and in real river water samples were in excellent agreement with the expected values.

Electrochemical Immunosensing of Cortisol in an Automated Microfluidic System Towards Point-of-Care Applications

This dissertation describes the development of a label-free, electrochemical immunosensing platform integrated into a low-cost microfluidic system for the sensitive, selective and accurate detection of cortisol, a steroid hormone co-related with many physiological disorders. Abnormal levels of cortisol is indicative of conditions such as Cushing’s syndrome, Addison’s disease, adrenal insufficiencies and more recently post-traumatic stress disorder (PTSD). Electrochemical detection of immuno-complex formation is utilized for the sensitive detection of Cortisol using Anti-Cortisol antibodies immobilized on sensing electrodes. Electrochemical detection techniques such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) have been utilized for the characterization and sensing of the label-free detection of Cortisol. The utilization of nanomaterial’s as the immobilizing matrix for Anti-cortisol antibodies that leads to improved sensor response has been explored. A hybrid nano-composite of Polyanaline-Ag/AgO film has been fabricated onto Au substrate using electrophoretic deposition for the preparation of electrochemical immunosening of cortisol. Using a conventional 3-electrode electrochemical cell, a linear sensing range of 1pM to 1µM at a sensitivity of 66µA/M and detection limit of 0.64pg/mL has been demonstrated for detection of cortisol. Alternately, a self-assembled monolayer (SAM) of dithiobis(succinimidylpropionte) (DTSP) has been fabricated for the modification of sensing electrode to immobilize with Anti-Cortisol antibodies. To increase the sensitivity at lower detection limit and to develop a point-of-care sensing platform, the DTSP-SAM has been fabricated on micromachined interdigitated microelectrodes (µIDE). Detection of cortisol is demonstrated at a sensitivity of 20.7µA/M and detection limit of 10pg/mL for a linear sensing range of 10pM to 200nM using the µIDE’s. A simple, low-cost microfluidic system is designed using low-temperature co-fired ceramics (LTCC) technology for the integration of the electrochemical cortisol immunosensor and automation of the immunoassay. For the first time, the non-specific adsorption of analyte on LTCC has been characterized for microfluidic applications. The design, fabrication technique and fluidic characterization of the immunoassay are presented. The DTSP-SAM based electrochemical immunosensor on µIDE is integrated into the LTCC microfluidic system and cortisol detection is achieved in the microfluidic system in a fully automated assay. The fully automated microfluidic immunosensor hold great promise for accurate, sensitive detection of cortisol in point-of-care applications.

An investigation into the suitability of time-of-flight mass spectrometry in forensic toxicology

Liquid chromatography coupled with mass spectrometry is one of the most powerful tools in the toxicologist’s arsenal to detect a wide variety of compounds from many different matrices. However, the huge number of potentially abused substances and new substances especially designed as intoxicants poses a problem in a forensic toxicology setting. Most methods are targeted and designed to cover a very specific drug or group of drugs while many other substances remain undetected. High resolution mass spectrometry, more specifically time-of-flight mass spectrometry, represents an extremely powerful tool in analysing a multitude of compounds not only simultaneously but also retroactively. The data obtained through the time-of-flight instrument contains all compounds made available from sample extraction and chromatography, which can be processed at a later time with an improved library to detect previously unrecognised compounds without having to analyse the respective sample again. The aim of this project was to determine the utility and limitations of time-of-flight mass spectrometry as a general and easily expandable screening method. The resolution of time-of-flight mass spectrometry allows for the separation of compounds with the same nominal mass but distinct exact masses without the need to separate them chromatographically. To simulate the wide variety of potentially encountered drugs in such a general screening method, seven drugs (morphine, cocaine, zolpidem, diazepam, amphetamine, MDEA and THC) were chosen to represent this variety in terms of mass, properties and functional groups. Consequently, several liquid-liquid and solid phase extractions were applied to urine samples to determine the most general suitable and unspecific extraction. Chromatography was optimised by investigating the parameters pH, concentration, organic solvent and gradient of the mobile phase to improve data obtained by the time-of-flight instrument. The resulting method was validated as a qualitative confirmation/identification method. Data processing was automated using the software TargetAnalysis, which provides excellent analyte recognition according to retention time, exact mass and isotope pattern. The recognition of isotope patterns allows excellent recognition of analytes even in interference rich mass spectra and proved to be a good positive indicator. Finally, the validated method was applied to samples received from the A& E Department of Glasgow Royal Infirmary in suspected drug abuse cases and samples received from the Scottish Prison Service, which we received from their own prevalence study targeting drugs of abuse in the prison population. The obtained data was processed with a library established in the course of this work.

Avaliação da eficiência de remoção do contaminante de origem farmacêutica atenolol pela planta phragmites australis em leitos construídos de macrófitas

O atenolol é um fármaco β-bloqueador normalmente encontrado em águas residuais devido à incapacidade que os processos convencionais de tratamento destas águas têm em removê-lo. Neste trabalho foram utilizados microcosmos de leitos construídos de macrófitas de fluxo sub-superficial utilizando uma matriz de argila expandida (LECA) e plantados com Phragmites australis para avaliar a sua capacidade em remover atenolol das águas residuais. Para a detecção e quantificação do atenolol em soluções aquosas (águas e efluentes) desenvolveu-se e optimizou-se uma metodologia analítica usando separação cromatográfica por HPLC e detecção espectrofotométrica por diode array (HPLC-DAD) ou por ultravioleta visível (HPLC-UV-Vis). Desenvolveu-se também um procedimento de limpeza e concentração de amostra por extracção em fase sólida (SPE), o qual foi utilizado sempre que as concentrações do analito se encontraram abaixo dos limites de quantificação do equipamento. A utilização desta metodologia de HPLC, combinada com uma eficaz pré-concentração por SPE, resultou num método analítico com um limite de quantificação muito reduzido (9 ngmL-1) e elevada reprodutibilidade (RSD<4%). A eficiência de remoção de atenolol pelos sistemas de macrófitas estudados foi de 93% após um tempo de retenção de 4 dias. Foram testados leitos só com LECA e com LECA e plantas para remoção do atenolol. Nos leitos só com LECA, a cinética de remoção foi caracterizada por um rápido passo inicial (uma remoção de aproximadamente 75% após apenas 24 h), o qual é frequentemente atribuído à adsorção na matriz de LECA. A remoção de atenolol nos leitos de LECA continuou a aumentar de forma constante até ao final do ensaio (8 dias), sendo, contudo cerca de 5-10% mais baixo do que o valor observado nos leitos das plantas após os 4 primeiros dias. Para o tempo de retenção de 4 dias a maioria do atenolol é removido pela matriz de LECA, porém um acréscimo de cerca de 12-14% relativamente à eficiência de remoção global pode ser atribuído às plantas (Phragmites australis), o que está de acordo com trabalhos anteriormente publicados. Apesar de ser necessário realizar mais testes utilizando sistemas em larga escala, de modo a conseguir avaliar totalmente o comportamento do atenolol num sistema de leitos construídos de macrófitas, o presente estudo apresenta a possibilidade de aplicar este tipo de sistemas, relativamente baratos, no tratamento de águas residuais contaminadas com atenolol. ABSTRACT: Atenolol is a β-blocker drug commonly found in wastewaters due to the inability of the conventional wastewater treatment processes to remove it. ln this study, subsurface flow constructed wetland microscosm systems have been established with a matrix of light expanded clay aggregates (LECA) and planted with Phragmites australis in order to evaluate their ability to remove atenolol from wastewater. For the detection and quantification of atenolol in aqueous solutions (water and wastewater), an adequate analytical methodology was developed and optimized using chromatographic separation by HPLC and diode array (DAD) or UV-Vis spectrophotometric detection. A sample clean-up and preconcentration procedure by solid phase extraction (SPE) was also developed for use whenever the concentration levels of the analyte were below the instrument's limit of quantification. Combined with an efficient SPE concentration step, the use of HPLC yielded an analytical method for atenolol quantification with very low LOQ (9 ngmL-1) and high reproducibility (RSD< 4%). Overall atenolol removal efficiency of 93% was achieved after a retention time of only 4 days with the microcosm systems planted with Phragmites australis. The removal kinetics was characterized by an initial fast step (removal of about 75% after just 24h) which is mainly attributable to adsorption on the LECA matrix. Atenolol removal in LECA beds continues to increase in a steady pace up to the end of the assay (8 days) being nevertheless about 5-l 0% lower than those observed in the planted beds after the first 4 days. For the retention time of 4 days most of the atenolol is removed by the LECA matrix but an additional 12-14% to the overall removal efficiency can be attributed to the Phragmites plants, which comes in agreement with other published reports. Despite the fact that further tests using larger­ scale systems are required to fully evaluate the atenolol behavior in a constructed wetland system, this study points out to the possible application of these low-cost wastewater systems to treat atenolol contaminated wastewater.