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The present work reports the results concerning the development and implementation of the first electrochemical biosensor for acrylamide determination, based on a direct biochemical interaction between the analyte and intact bacterial cells, with intracellular enzymatic activity. The biological recognition element consisted of whole cells of Pseudomonas aeruginosa containing intracellular amidase activity, which catalyses the hydrolysis of acrylamide producing ammonium ion (NH 4 +) and acrylic acid. The transduction process was accomplished by means of an ammonium ion selective electrode. Whole cells were firstly immobilized on single discs of polymeric membranes, such as polyethersulphone, nylon and polycarbonate, which were, then, attached to the surface of the selective electrode. However, it was observed a significant loss of cells each time the biosensor was used, namely at the beginning of the assay, when the membranes were attached to the ammonium electrode, and after the assay, when removed for storage purposes. This evidence determined a premature decrease in the biosensor's stability. Instead of using single membrane discs, a "sandwich" design, with two membrane discs was considered. This way the cells remain contained between the membranes, never contacting the electrode's surface, preventing their premature loss. Consequently, the activity of the biosensor could be maintained for longer periods of time. The analytical performance of the biosensor was evaluated. The best results were obtained when polyethersulphone double membranes were used. A typical response of 120 mV (after 6 min reaction time), a Nernstian slope of 48 mV/decade, a limit of detection of 6.31×10-4 M and a half-life time of 27 days, are examples of some figures of merit observed for this biosensor.

foi utilizada na determinação simultânea de quantidades traço dos metais tóxicos Pb(II), Cd(II) e Cu(II) em folhas de choupo (populus), utilizadas como bio-indicador, recolhidas numa área de tráfego automóvel intenso da cidade de Lisboa. As folhas, após secagem, foram submetidas a um processo de digestão ácida por microondas. Para a aplicação desta técnica à análise das folhas foi realizado um estudo de optimização dos vários parâmetros voltamétricos. O eletrodo de trabalho consistiu num filme de mercúrio depositado numa superfície de carbono vítreo. O sistema Ag/AgCl foi utilizado como referência e um fio de Platina como eletrodo auxiliar. Concentrações médias (em mg de metal/kg de matéria seca -folhas) de 2,6, 0,18 e 5,0 foram obtidas para o Pb(II), o Cd(II) e o Cu(II), respectivamente. O valor obtido para o chumbo coincide com o obtido pelo método de referência baseado na Espectrofotometria de Absorção Atômica em Forno de Grafite (EAAFG).Square wave anodic stripping voltammetry (SWASV) was applied to the simultaneous determination of trace amounts of toxic metals Pb(II), Cd(II) and Cu(II) in white poplar (populus) leaves, used as bio indicator and gathered in a chosen area of the city of Lisbon with very high traffic intensity. The leaves were dried and subsequently exposed to an acid digestion microwave process. Square wave parameters were optimized for the voltammetric analysis of the samples. The working electrode consisted of a thin mercury film (TMFE) deposited on the surface of vitreous carbon. The pair Ag/AgCl was used as the reference electrode and a Pt wire as the auxiliary electrode. Average concentrations (in mg of metal/kg of dry matter-leaves) of 2.6, 0.18, and 5.0 were obtained for Pb(II), Cd(II) and Cu(II), respectively. The value for lead coincides with the one obtained by the reference method based on Graphite Furnace Atomic Absorption Spectrophotometry (GFAAS).

Acrylamide is a toxic amide with potentially hazardous effects on the environment and human health. This paper reports the results regarding the development of a potentiometric biosensor in order to determine the amount of this amide in wastewater samples. The biosystem consisted of whole cells of Pseudomonas aeruginosa containing intracellular amidase activity which hydrolyses acrylamide producing ammonium ion and acrylic acid. The cells were immobilized on the surface of several types of membranes such as polyethersulfone, nitrocellulose and nylon, in the presence of glutaraldehyde as bifunctional reagent, and then attached to the surface of an ammonium ion selective electrode. Polyethersulfone was revealed to be the most adequate in terms of biosensor response. The effect of glutaraldehyde concentration was also studied and 5% (v/v) was chosen as the optimum concentration value. The results obtained revealed excellent analytical characteristics of the biosensor such as good linear response in the range of 0.5 to 100mM of acrylamide, a detection limit of 4.48 x 10 -5 M, a response time of 55 s, a sensitivity of 58.9mV/mM. This system was also tested in real samples of complex matrix, namely wastewater from an industrial plant where an average substrate recover of 93.3% was obtained.

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Pseudomonas aeruginosa (P. aeruginosa) possesses intracellular amidase activity, which catalyses the hydrolysis of short aliphatic amides producing NH4+, and has already been used along with an ammonium ion selective electrode for amide quantification. However, the incorporation of a biological membrane turned to be a challenging process and either the final arrangement was prone to amidase losses or the recovery of the sensor coating after the interaction took too long. In this article a flow injection system with an ammonium acoustic wave sensor is proposed, and after testing several different arrangements for the biological element, the ultimate choice consisted of the immobilization of a P. aeruginosa cell-free extract in the inner wall of a tubular glass reactor, which resulted in a reliable analytical system. Response times less than one minute and complete recovery in less than two minutes assured conveniently fast analysis. The analytical system, as long as the column was properly stored in HEPES buffer containing 2 mM β-mercaptoethanol and 1 mM benzamidine and refrigerated when not in use, could be used at least for 20 working days, along a period of one month, maintaining the initial sensitivity.

Pseudomonas aeruginosa (P. aeruginosa) is a Gram-negative bacterium quite versatile that grows in the soil, in coastal marine habitats, as well as in the tissues of plants and animals. P. aeruginosa is the source of amidase (acylamide amidohydrolase E.C. 3.5.1.4) which catalyzes the hydrolysis of a small range of short aliphatic amides into the corresponding carboxylic acids and ammonia.A low cost piezoelectric quartz crystal coated with a selective membrane for ammonium was used to detect the reaction product.Conversion of amide into the correspondent amine was achieved both with cell-free extract of P. Aeruginosa or the whole cells. This conversion was first performed in batch and later on injected into the sensor system where a buffer carrier was flowing over the coated crystal. Another approach consisted in incorporating a conversion reactor with the immobilized cell-free extract of P. Aeruginosa in the FIA system. Amide solutions were injected and carried by the buffer stream through the reactor and then directed to the sensor. Different supports were used for immobilization, such as calcium alginate beads, glass beads and the inside walls of a hollow glass column.The best arrangement allowed acetamide determination without sensitivity lost for 1-month period.