ß-lactamases in the biochemistry and molecular biology laboratory

β-lactamases are hydrolytic enzymes that inactivate the β-lactam ring of antibiotics such as penicillins and cephalosporins. The major diversity of studies carried out until now have mainly focused on the characterization of β-lactamases recovered among clinical isolates of Gram-positive staphylococci and Gram-negative enterobacteria, amongst others. However, only some studies refer to the detection and development of β-lactamases carriers in healthy humans, sick animals, or even in strains isolated from environmental stocks such as food, water, or soils. Considering this, we proposed a 10-week laboratory programme for the Biochemistry and Molecular Biology laboratory for majors in the health, environmental, and agronomical sciences. During those weeks, students would be dealing with some basic techniques such as DNA extraction, bacterial transformation, polymerase chain reaction (PCR), gel electrophoresis, and the use of several bioinformatics tools. These laboratory exercises would be conducted as a mini research project in which all the classes would be connected with the previous ones. This curriculum was compared in an experiment involving two groups of students from two different majors. The new curriculum, with classes linked together as a mini research project, was taught to a major in Pharmacy and an old curriculum was taught to students from environmental health. The results showed that students who were enrolled in the new curriculum obtained better results in the final exam than the students who were enrolled in the former curriculum. Likewise, these students were found to be more enthusiastic during the laboratory classes than those from the former curriculum.

Oriented Tailoring of Plastic Antibodies for Prostate Specific Antigen and Application of the Imprinted Material as Ionophore in Potentiometric Detection

Prostate Specific Antigen (PSA) is the biomarker of choice for screening prostate cancer throughout the population, with PSA values above 10 ng/mL pointing out a high probability of associated cancer1. According to the most recent World Health Organization (WHO) data, prostate cancer is the commonest form of cancer in men in Europe2. Early detection of prostate cancer is thus very important and is currently made by screening PSA in men over 45 years old, combined with other alterations in serum and urine parameters. PSA is a glycoprotein with a molecular mass of approximately 32 kDa consisting of one polypeptide chain, which is produced by the secretory epithelium of human prostate. Currently, the standard methods available for PSA screening are immunoassays like Enzyme-Linked Immunoabsorbent Assay (ELISA). These methods are highly sensitive and specific for the detection of PSA, but they require expensive laboratory facilities and high qualify personal resources. Other highly sensitive and specific methods for the detection of PSA have also become available and are in its majority immunobiosensors1,3-5, relying on antibodies. Less expensive methods producing quicker responses are thus needed, which may be achieved by synthesizing artificial antibodies by means of molecular imprinting techniques. These should also be coupled to simple and low cost devices, such as those of the potentiometric kind, one approach that has been proven successful6. Potentiometric sensors offer the advantage of selectivity and portability for use in point-of-care and have been widely recognized as potential analytical tools in this field. The inherent method is simple, precise, accurate and inexpensive regarding reagent consumption and equipment involved. Thus, this work proposes a new plastic antibody for PSA, designed over the surface of graphene layers extracted from graphite. Charged monomers were used to enable an oriented tailoring of the PSA rebinding sites. Uncharged monomers were used as control. These materials were used as ionophores in conventional solid-contact graphite electrodes. The obtained results showed that the imprinted materials displayed a selective response to PSA. The electrodes with charged monomers showed a more stable and sensitive response, with an average slope of -44.2 mV/decade and a detection limit of 5.8X10-11 mol/L (2 ng/mL). The corresponding non-imprinted sensors showed smaller sensitivity, with average slopes of -24.8 mV/decade. The best sensors were successfully applied to the analysis of serum samples, with percentage recoveries of 106.5% and relatives errors of 6.5%.

Determinação de autoanticorpos em doentes com suspeita de hepatite autoimune e cirrose biliar primária

O diagnóstico de doença hepática autoimune em doentes com patologia hepática implica a exclusão de outras causas de lesão hepática como vírica, alcoólica, tóxica, devido a alterações genéticas ou metabólicas, esteatose hepática não alcoólica e uma criteriosa avaliação de dados clínicos, bioquímicos, histológicos e colangiográficos especificas destas patologias (Invernizzi et al 2007) O diagnóstico e tratamento precoces destas patologias são fundamentais para a prevenção da alta morbilidade e mortalidade associada a estes doentes. O despiste de patologia hepática autoimune assenta na utilização de testes serológicos para a deteção de autoanticorpos associados a estas patologias. O conhecimento destes testes e a interpretação dos resultados obtidos revelam-se fundamentais para o diagnóstico ou exclusão destas doenças (Beuers 2005). Deste modo, foi objetivo deste trabalho a pesquisa e identificação de autoanticorpos em uso clínico: ANA, AMA, AML, ANCA, Anti-SLA/LP, anti-LKM, anti-LC1 e anti-actina F, em doentes com suspeita de HAI e CBP em que foi excluída causa vírica, alcoólica e tóxica. O trabalho incidiu particularmente na comparação dos resultados do perfil de autoanticorpos de pedidos feitos ao exterior com os resultados obtidos recorrendo à utilização de um novo kit de imunoblot, e assim determinar a relevância da introdução da pesquisa dos novos autoanticorpos e avaliar a relação custo/benefício da implementação do kit BlueDot liver da D-tek® na rotina laboratorial do serviço de Patologia Clínica do Hospital Pedro Hispano. Os resultados encontrados foram de 100% de concordância entre os métodos de imunofluorescência indireta e imunoblot, e Elisa e Imunoblot. Deste modo seria uma boa estratégia a implementação desta última técnica na rotina laboratorial uma vez que proporciona uma rápida disponibilização dos resultados para o clínico, antecipando desta forma o diagnóstico e o início rápido do tratamento em benefício do doente. Por outro lado, quando analisámos a relação custo/beneficio, seria vantajosa a implementação desta técnica uma vez que o laboratório dispõe de capacidade técnica, e o custo de aquisição do kit não excede o valor praticado atualmente correspondendo a uma poupança de 51%.

Celiac disease detection using a transglutaminase electrochemical immunosensor fabricated on nanohybrid screen-printed carbon electrodes

Celiac disease is a gluten-induced autoimmune enteropathy characterized by the presence of tissue tranglutaminase (tTG) autoantibodies. A disposable electrochemical immunosensor (EI) for the detection of IgA and IgG type anti-tTG autoantibodies in real patient’s samples is presented. Screen-printed carbon electrodes (SPCE) nanostructurized with carbon nanotubes and gold nanoparticles were used as the transducer surface. This transducer exhibits the excellent characteristics of carbon–metal nanoparticle hybrid conjugation and led to the amplification of the immunological interaction. The immunosensing strategy consisted of the immobilization of tTG on the nanostructured electrode surface followed by the electrochemical detection of the autoantibodies present in the samples using an alkaline phosphatase (AP) labelled anti-human IgA or IgG antibody. The analytical signal was based on the anodic redissolution of enzymatically generated silver by cyclic voltammetry. The results obtained were corroborated with a commercial ELISA kit indicating that the electrochemical immunosensor is a trustful analytical screening tool.

Molecular imprinted nanoelectrodes for ultra sensitive detection of ovarian cancer marker

The relentless discovery of cancer biomarkers demands improved methods for their detection. In this work, we developed protein imprinted polymer on three-dimensional gold nanoelectrode ensemble (GNEE) to detect epithelial ovarian cancer antigen-125 (CA 125), a protein biomarker associated with ovarian cancer. CA 125 is the standard tumor marker used to follow women during or after treatment for epithelial ovarian cancer. The template protein CA 125 was initially incorporated into the thin-film coating and, upon extraction of protein from the accessible surfaces on the thin film, imprints for CA 125 were formed. The fabrication and analysis of the CA 125 imprinted GNEE was done by using cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) techniques. The surfaces of the very thin, protein imprinted sites on GNEE are utilized for immunospecific capture of CA 125 molecules, and the mass of bound on the electrode surface can be detected as a reduction in the faradic current from the redox marker. Under optimal conditions, the developed sensor showed good increments at the studied concentration range of 0.5–400 U mL−1. The lowest detection limit was found to be 0.5 U mL−1. Spiked human blood serum and unknown real serum samples were analyzed. The presence of non-specific proteins in the serum did not significantly affect the sensitivity of our assay. Molecular imprinting using synthetic polymers and nanomaterials provides an alternative approach to the trace detection of biomarker proteins.

Ultrasensitive detection of ovarian cancer marker using immunoliposomes and gold nanoelectrodes

Mucin-16 (MUC16) is the established ovarian cancer marker used to follow the disease during or after treatment for epithelial ovarian cancer. The emerging science of cancer markers also demands for the new sensitive detection methods. In this work, we have developed an electrochemical immunosensor for antigen MUC16 using gold nanoelectrode ensemble (GNEE) and ferrocene carboxylic acid encapsulated liposomes tethered with monoclonal anti-Mucin-16 antibodies ( MUC16). GNEEs were fabricated by electroless deposition of the gold within the pores of polycarbonate track-etched membranes. Afterwards, MUC16 were immobilized on preformed self-assembled monolayer of cysteamine on the GNEE via cross-linking with EDC-Sulfo-NHS. A sandwich immunoassay was performed on MUC16 functionalized GNEE with MUC16 and immunoliposomes. The differential pulse voltammetry was employed to quantify the faradic redox response of ferrocene carboxylic acid released from immunoliposomes. The dose–response curve for MUC16 concentration was found between the range of 0.001–300 U mL−1. The lowest detection limit was found to be 5 × 10−4 U mL−1 (S/N = 3). We evaluated the performance of this developed immunosensor with commercial ELISA assay by comparing results obtained from spiked serum samples and real blood serum samples from volunteers.

Electrochemical immunosensor for multiplexed detection of food-borne pathogens using nanocrystal bioconjugates and MWCNT screen-printed electrode

Bacterial food poisoning is an ever-present threat that can be prevented with proper care and handling of food products. A disposable electrochemical immunosensor for the simultaneous measurements of common food pathogenic bacteria namely Escherichia coli O157:H7 (E. coli), campylobacter and salmonella were developed. The immunosensor was fabricated by immobilizing the mixture of anti-E. coli, anticampylobacter and anti-salmonella antibodies with a ratio of 1:1:1 on the surface of the multiwall carbon nanotube-polyallylamine modified screen printed electrode (MWCNT-PAH/SPE). Bacteria suspension became attached to the immobilized antibodies when the immunosensor was incubated in liquid samples. The sandwich immunoassay was performed with three antibodies conjugated with specific nanocrystal ( -E. coli-CdS, -campylobacter-PbS and -salmonella-CuS) which has releasable metal ions for electrochemical measurements. The square wave anodic stripping voltammetry (SWASV) was employed to measure released metal ions from bound antibody nanocrystal conjugates. The calibration curves for three selected bacteria were found in the range of 1 × 103 – 5 × 105 cells mL−1 with the limit of detection (LOD) 400 cells mL−1 for salmonella, 400 cells mL−1 for campylobacter and 800 cells mL−1 for E. coli. The precision and sensitivity of this method show the feasibility of multiplexed determination of bacteria in milk samples.

Enzymatic determination of choline in milk using a FIA system with potentiometric detection

The development of a FIA system for the determination of total choline content in several types of milk is described. The samples were submitted to hydrochloric acid digestion before injection into the system and passed through an enzymatic reactor containing choline oxidase immobilised on glass beads. This enzymatic reaction releases hydrogen peroxide which then reacts with a solution of iodide. The decrease in the concentration of iodide ion is quantified using an iodide ion selective tubular electrode based on a homogeneous crystalline membrane. Validation of the results obtained with this system was performed by comparison with results from a method described in the literature and applied to the determination of total choline in milks. The relative deviation was always < 5%. The repeatability of the method developed was assessed by calculation of the relative standard deviation (RSD) for 12 consecutive injections of one sample. The RSD obtained was < 0.6%.

Sensors for the detection and quantification of bacterial contamination in water for human use

The deterioration of water quality by Cyanobacteria cause outbreaks and epidemics associated with harmful diseases in Humans and animals because of the toxins that they release. Microcystin-LR is one of the hepatotoxins most widely studied and the World Health Organization, recommend a maximum value of 1mgL 1 in drinking water. Highly specific recognition molecules, such as molecular imprinted polymers are developed to quantify microcystins in waters for human use and shown to be of great potential in the analysis of these kinds of samples. The obtained results were auspicious, the detection limit found, 1.5mgL 1, being of the same order of magnitude as the guideline limit recommended by the WHO. This technology is very promising because the sensors are stable and specific, and the technology is inexpensive and allows for rapid on-site monitoring.

A multiresidue method for the analysis of carbamate and urea pesticides from soils by microwave-assisted extraction and liquid chromatography with photodiode array detection

A multiresidue approach using microwave-assisted extraction and liquid chromatography with photodiode array detection was investigated for the determination of butylate, carbaryl, carbofuran, chlorpropham, ethiofencarb, linuron,metobromuron, and monolinuron in soils. The critical parameters of the developed methodology were studied. Method validation was performed by analyzing freshly and aged spiked soil samples. The recoveries and relative standard deviations reached using the optimized conditions were between 77.0 ± 0.46% and 120 ± 2.9% except for ethiofencarb (46.4 ± 4.4% to 105 ± 1.6%) and butylate (22.1 ± 7.6% to 49.2 ± 11%). Soil samples from five locations of Portugal were analysed.

Square-wave adsorptive-stripping voltammetric detection in the quality control of fluoxetine

Electroanalytical methods based on square-wave adsorptive-stripping voltammetry (SWAdSV) and flow-injection analysis with square-wave adsorptive-stripping voltammetric detection (FIA-SWAdSV) were developed for the determination of fluoxetine (FXT). The methods were based on the reduction of FXT at a mercury drop electrode at -1.2 V versus Ag/AgCl, in a phosphate buffer of pH 12.0, and on the possibility of accumulating the compound at the electrode surface. The SWAdSV method was successfully applied in the quantification of FXT in pharmaceutical products, human serum samples, and in drug dissolution studies. Because the presence of dissolved oxygen did not interfere significantly with the analysis, it was possible to quantify FXT in several pharmaceutical products using FIA-SWAdSV. This method enables analysis of up to 120 samples per hour at reduced costs.

Development of an FIA system with amperometric detection for determination of bentazone in estuarine waters

On the basis of its electrochemical behaviour a new flow-injection analysis (FIA) method with amperometric detection has been developed for quantification of the herbicide bentazone (BTZ) in estuarine waters. Standard solutions and samples (200 µL) were injected into a water carrier stream and both pH and ionic strength were automatically adjusted inside the manifold. Optimization of critical FIA conditions indicated that the best analytical results were obtained at an oxidation potential of 1.10 V, pH 4.5, and an overall flow-rate of 2.4 mL min–1. Analysis of real samples was performed by means of calibration curves over the concentration range 2.5x10–6 to 5.0x10–5 mol L–1, and results were compared with those obtained by use of an independent method (HPLC). The accuracy of the amperometric determinations was ascertained; errors relative to the comparison method were below 4% and sampling rates were approximately 100 samples h–1. The repeatability of the proposed method was calculated by assessing the relative standard deviation (%) of ten consecutive determinations of one sample; the value obtained was 2.1%.

Maltol determination in food by microwave assisted extraction and electrochemical detection

An electrochemical method is proposed for the determination of maltol in food. Microwave-assisted extraction procedures were developed to assist sample pre-treating steps. Experiments carried out in cyclic voltammetry showed an irreversible and adsorption controlled reduction of maltol. A cathodic peak was observed at -1.0 V for a Hanging Mercury Drop Electrode versus an AgCl/Ag (in saturated KCl), and the peak potential was pH independent. Square wave voltammetric procedures were selected to plot calibration curves. These procedures were carried out with the optimum conditions: pH 6.5; frequency 50 Hz; deposition potential 0.6 V; and deposition time 10 s. A linear behaviour was observed within 5.0 × 10-8 and 3.5 × 10-7 M. The proposed method was applied to the analysis of cakes, and results were compared with those obtained by an independent method. The voltammetric procedure was proven suitable for the analysis of cakes and provided environmental and economical advantages, including reduced toxicity and volume of effluents and decreased consumption of reagents.

Screening of grapes and wine for azoxystrobin, kresoxim-methyl and trifloxystrobin fungicides by HPLC with diode array detection

The Quinone outside Inhibitors (QoI) are one of the most important and recent fungicide groups used in viticulture and also allowed by Integrated Pest Management. Azoxystrobin, kresoxim-methyl and trifloxystrobin are the main active ingredients for treating downy and powdery mildews that can be present in grapes and wines. In this paper, a method is reported for the analysis of these three QoI-fungicides in grapes and wine. After liquid–liquid extraction and a clean-up on commercial silica cartridges, analysis was by isocratic HPLC with diode array detection (DAD) with a run time of 13 min. Confirmation was by solid-phase micro-extraction (SPME), followed by GC/MS determination. The main validation parameters for the three compounds in grapes and wine were a limit of detection up to 0.073mg kg-1, a precision not exceeding 10.0% and an average recovery of 93% ±38.

Multiplexed electrochemical immunosensor for detection of celiac disease serological markers

Celiac disease (CD) is a gluten-induced autoimmune enteropathy characterized by the presence of antibodies against gliadin (AGA) and anti-tissue transglutaminase (anti-tTG) antibodies. A disposable electrochemical dual immunosensor for the simultaneous detection of IgA and IgG type AGA and antitTG antibodies in real patient’s samples is presented. The proposed immunosensor is based on a dual screen-printed carbon electrode, with two working electrodes, nanostructured with a carbon–metal hybrid system that worked as the transducer surface. The immunosensing strategy consisted of the immobilization of gliadin and tTG (i.e. CD specific antigens) on the nanostructured electrode surface. The electrochemical detection of the human antibodies present in the assayed serum samples was carried out through the antigen–antibody interaction and recorded using alkaline phosphatase labelled anti-human antibodies and a mixture of 3-indoxyl phosphate with silver ions was used as the substrate. The analytical signal was based on the anodic redissolution of enzymatically generated silver by cyclic voltammetry. The results obtained were corroborated with commercial ELISA kits indicating that the developed sensor can be a good alternative to the traditional methods allowing a decentralization of the analyses towards a point-of-care strategy.