Surface Imprinting Approach on Screen Printed Electrodes Coated with Carboxylated PVC for Myoglobin detection with Electrochemical Transduction

A novel surface molecularly-imprinted (MI) material to detect myoglobin (Myo) using gold screen printed electrodes (SPE) was developed. The sensitive detection was carry out by introducing a carboxylic polyvinyl chloride (PVC-COOH) layer on gold SPE surface. Myo was attached to the surface of gold SPE/PVC-COOH and the vacant spaces around it were filled by polymerizing acrylamide and N,N-methylenebisacrylamide (cross-linker). This polymerization was initiated by ammonium persulphate. After removing the template, the obtained material was able to rebind Myo and discriminate it among other interfering species. Various characterization techniques including electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) confirmed the surface modification. This sensor seemed a promising tool for screening Myo in point-of-care.

Determination of Microcystin-LR in waters in the subnanomolar range by sol–gel imprinted polymers on solid contact electrodes

The present work reports new sensors for the direct determination of Microcystin-LR (MC-LR) in environmental waters. Both selective membrane and solid contact were optimized to ensure suitable analytical features in potentiometric transduction. The sensing layer consisted of Imprinted Sol–Gel (ISG) materials capable of establishing surface interactions with MC-LR. Non-Imprinted Sol–Gel (NISG) membranes were used as negative control. The effects of an ionic lipophilic additive, time of sol–gel polymerization, time of extraction of MC-LR from the sensitive layer, and pH were also studied. The solid contact was made of carbon, aluminium, titanium, copper or nickel/chromium alloys (80 : 20 or 90 : 10). The best ISG sensor had a carbon solid contact and displayed average slopes of 211.3 mV per decade, with detection limits of 7.3 1010 M, corresponding to 0.75 mg L1 . It showed linear responses in the range of 7.7 1010 to 1.9 109 M of MC-LR (corresponding to 0.77–2.00 mg L1 ), thus including the limiting value for MC-LR in waters (1.0 mg L1 ). The potentiometric-selectivity coefficients were assessed by the matched potential method for ionic species regularly found in waters up to their limiting levels. Chloride (Cl) showed limited interference while aluminium (Al3+), ammonium (NH4 + ), magnesium (Mg2+), manganese (Mn2+), sodium (Na+ ), and sulfate (SO4 2) were unable to cause the required potential change. Spiked solutions were tested with the proposed sensor. The relative errors and standard deviation obtained confirmed the accuracy and precision of the method. It also offered the advantages of low cost, portability, easy operation and suitability for adaptation to flow methods.

Artificial antibodies for troponin T by its imprinting on the surface of multiwalled carbon nanotubes: Its use as sensory surfaces

A novel artificial antibody for troponin T (TnT) was synthesized by molecular imprint (MI) on the surface of multiwalled carbon nanotubes (MWCNT). This was done by attaching TnT to the MWCNT surface, and filling the vacant spaces by polymerizing under mild conditions acrylamide (monomer) in N,N′-methylenebisacrylamide (cross-linker) and ammonium persulphate (initiator). After removing the template, the obtained biomaterial was able to rebind TnT and discriminate it among other interfering species. Stereochemical recognition of TnT was confirmed by the non-rebinding ability displayed by non-imprinted (NI) materials, obtained by imprinting without a template. SEM and FTIR analysis confirmed the surface modification of the MWCNT. The ability of this biomaterial to rebind TnT was confirmed by including it as electroactive compound in a PVC/plasticizer mixture coating a wire of silver, gold or titanium. Anionic slopes of 50 mV decade−1 were obtained for the gold wire coated with MI-based membranes dipped in HEPES buffer of pH 7. The limit of detection was 0.16 μg mL−1. Neither the NI-MWCNT nor the MWCNT showed the ability to recognize the template. Good selectivity was observed against creatinine, sucrose, fructose, myoglobin, sodium glutamate, thiamine and urea. The sensor was tested successfully on serum samples. It is expected that this work opens new horizons on the design of new artificial antibodies for complex protein structures.

Ciprofloxacin-imprinted polymeric receptors as ionophores for potentiometric transduction

A 3D-mirror synthetic receptor for ciprofloxacin host–guest interactions and potentiometric transduction is presented. The host cavity was shaped on a polymeric surface assembled with methacrylic acid or 2-vinyl pyridine monomers by radical polymerization. Molecularly imprinted particles were dispersed in 2-nitrophenyl octyl ether and entrapped in a poly(vinyl chloride) matrix. The sensors exhibited a near-Nernstian response in steady state evaluations. Slopes and detection limits ranged from 26.8 to 50.0 mV decade−1 and 1.0 × 10−5 to 2.7 × 10−5 mol L−1, respectively. Good selectivity was observed for trimethoprim, enrofloxacin, tetracycline, cysteine, galactose, hydroxylamine, creatinine, ammonium chloride, sucrose, glucose, sulphamerazine and sulfadiazine. The sensors were successfully applied to the determination of ciprofloxacin concentrations in fish and in pharmaceuticals. The method presented offered the advantages of simplicity, accuracy, applicability to colored and turbid samples and automation feasibility, as well as confirming the use of molecularly imprinted polymers as ionophores for organic ion recognition in potentiometric transduction.

Trimethoprim-selective electrodes with molecularly imprinted polymers acting as ionophores and potentiometric transduction on graphite solid-contact

This work proposes a new biomimetic sensor material for trimethoprim. It is prepared by means of radical polymerization, having trimethylolpropane trimethacrylate as cross-linker, benzoyl peroxide as radicalar iniciator, chloroform as porogenic solvent, and methacrylic acid and 2-vinyl pyridine as monomers. Different percentages of sensor in a range between 1 and 6% were studied. Their behavior was compared to that obtained with ion-exchanger quaternary ammonium salt (additive tetrakis(p-chlorophenyl)borate or tetraphenylborate). The effect of an anionic additive in the sensing membrane was also tested. Trimethoprim sensors with 1% of imprinted particles from methacrylic acid monomers showed the best response in terms of slope (59.7 mV/decade) and detection limit (4.01 × 10− 7 mol/L). These electrodes displayed also a good selectivity towards nickel, manganese aluminium, ammonium, lead, potassium, sodium, iron, chromium, sulfadiazine, alanine, cysteine, tryptophan, valine and glycine. The sensors were not affected by pH changes from 2 to 6. They were successfully applied to the analysis of water from aquaculture.

Carnitine tailored Sensors on Surface Molecular Imprinting based on Graphene layers

III Jornadas de Electroquímica e Inovação (Electroquímica e Nanomateriais), na Universidade de Trás-os-Montes e Alto Douro, Vila Real, 16 a 17 de Setembro de 2013

Host-Tailored Sensors for Carnitine Potentiometric Measurements based on Surface Molecular Imprinting

Graduate Student Symposium on Molecular Imprinting 2013, na Queen’s University, Belfast, United Kingdom, 15 a 17 de Agosto de 2013

Measuring the cytochrome c nitrite reductase activity—practical considerations on the enzyme assays

Hindawi Publishing Corporation Bioinorganic Chemistry and Applications Volume 2010, Article ID 634597, 8 pages

Baseline Susceptibility of Primary HIV-2 to Entry Inhibitors

BACKGROUND: The baseline susceptibility of primary HIV-2 to maraviroc (MVC) and other entry inhibitors is currently unknown. METHODS: The susceptibility of 19 HIV-2 isolates obtained from asymptomatic and AIDS patients and seven HIV-1 clinical isolates to the fusion inhibitors enfuvirtide (ENF) and T-1249, and to the coreceptor antagonists AMD3100, TAK-779 and MVC, was measured using a TZM-bl cell-based assay. The 50% inhibitory concentration (IC(50)), 90% inhibitory concentration (IC(90)) and dose-response curve slopes were determined for each drug. RESULTS: ENF and T-1249 were significantly less active on HIV-2 than on HIV-1 (211- and 2-fold, respectively). AMD3100 and TAK-779 inhibited HIV-2 and HIV-1 CXCR4 tropic (X4) and CCR5 tropic (R5) variants with similar IC(50) and IC(90) values. MVC, however, inhibited the replication of R5 HIV-2 variants with significantly higher IC(90) values (42.7 versus 9.7 nM; P<0.0001) and lower slope values (0.7 versus 1.3; P<0.0001) than HIV-1. HIV-2 R5 variants derived from AIDS patients were significantly less sensitive to MVC than variants from asymptomatic patients, this being inversely correlated with the absolute number of CD4(+) T-cells. CONCLUSIONS: T-1249 is a potent inhibitor of HIV-2 replication indicating that new fusion inhibitors might be useful to treat HIV-2 infection. Coreceptor antagonists TAK-779 and AMD3100 are also potent inhibitors of HIV-2 replication. The reduced sensitivity of R5 variants to MVC, especially in severely immunodeficient patients, indicates that the treatment of HIV-2-infected patients with MVC might require higher dosages than those used in HIV-1 patients, and should be adjusted to the disease stage.

Cancer theranostics: multifunctional gold nanoparticles for diagnostics and therapy

Doctorate in Biology, Specialty in Biotechnology

Fate of nitrogen in bioreactor landfills: lab-scale in situ aeration in well decomposed MSW

Dissertação para obtenção do Grau de Mestre em Engenharia do ambiente, perfil de engenharia sanitária

Gold nanoparticles for plasmid delivery

Gene therapy presents an ideal strategy for the treatment of genetic as well as acquired diseases, such as cancer and typically involves the insertion of a functioning gene into cells to correct a cellular dysfunction or to provide a new cellular function. Gene delivery vectors are based in two models: viral and non-viral. Viral vectors have high transfection efficiency but their major barrier is immunogenicity. Since the non-viral vectors have no immunogenicity, these have been widely studied. Gold nanoparticles have been proposed as optimal delivery systems of genetic material, due their small size, high surface-to-volume ratio and the ability to be functionalized with multiple molecules. In the present work, an AuNP-based formulation was developed to deliver a plasmid in a colorectal cancer cell line, containing as reporter gene the gene encoding to EGFP. The delivery system resulted from the functionalization of 14 nm AuNP with a PEG layer (4300114 PEG chains/AuNP), which increases stability and biocompatibility of AuNPs; quaternary ammonium groups which provide positive charges that allow electrostatic binding of plasmid, which is considered the therapeutic agent to be transported into cells. The system developed was characterized by UV-vis spectroscopy, DLS, TEM and by electrophoretic mobility, yielding a formulation with 113.5 nm.Transfection efficiency of the formulation developed was evaluated through PCR and through EGFP expression by fluorescence microscopy and fluorescence spectroscopy. The internalization was observed 3h post transfection; however a low level of EGFP expression was achieved. After 24h of incubation, EGFP expression increases just 3 times compared to non-transfected cells. The commercial system (Lipofectamine) expressed EGFP 5 times more than the system developed AuNP@PEG@R4N+@pEGFP. This difference could be related to lower translocation to the nucleus.

Enrichment of a PHA producing microbial community in a continuous bioreactor setup

Polyhydroxyalkanoates (PHAs) are biosynthetic polyesters, biodegradable and biocompatible making them of great interest for industrial purposes. The use of low value substrates with mixed microbial communities (MMC) is a strategy currently used to decrease the elevated PHA production costs. PHA production process requires an important step for selection and enrichment of PHA-storing microorganisms which is usually carried out in a Sequencing Batch Reactor (SBR). The aim of this study was to optimize the PHA accumulating culture selection stage using a 2-stage Continuous Stirrer Tank Reactor (CSTR) system. The system was composed by two separate feast and famine bioreactors operated continuously, mimicking the feast and famine phases in a SBR system. Acetate was used as carbon source and biomass seed was highly enriched in Plasticicumulans acidivorans obtained from activated sludge. The system was operated under two different sets of conditions (setup 1 and 2), maintaining a system total retention time of 12 hours and an OLR of 2.25 Cmmol/L.h-1. An average PHB-content of 3.3 % wt was obtained in setup 1 and 4.8% wt in setup 2. Several other experiments were performed in order to better understand the continuous system behaviour, using biomass from the continuous system. With the fed-batch experiment a maximum of 8.1% PHB was stored and the maximum substrate uptake and specific growth rates obtained in the growth experiment (1.15 Cmol Cmol-1.h-1 and 0.53 Cmol Cmol-1.h-1) were close to the ones from continuous system (1.12 Cmol Cmol-1.h-1 and 0.59 Cmol Cmol-1.h-1). The microbial community was characterized trough microscopic visualization, Denaturing Gradient Gel Electrophoresis (DGGE) analysis and Fluorescent in situ hybridization (FISH). The last studied performed mimicked the continuous system by building up a SBR system with all the same operational conditions while adding an extra acetate dosage during the 12 h cycle, simulating the substrate passing from the feast to the famine reactors under continuous operation. It was shown that possibly the continuous system was not able to efficiently select for PHB storing organisms under the operational conditions imposed, although the selected culture was capable of consuming the substrate and grow fast. This main conclusion might have resulted from two major factors affecting the system performance: the ammonium concentration in the Feast reactor and the amount of substrate leaching from the Feast to the Famine reactor.

Analysis, testing and development of safe cleaning methods of rusted stone material

Three different treatments were applied on several specimens of dolomitic and calcitic marble, properly stained with rust to mimic real situations (the stone specimens were exposed to the natural environment for about six months in contact with rusted iron). Thirty six marble specimens, eighteen calcitic and eighteen dolomitic, were characterized before and after treatment and monitored throughout the cleaning tests. The specimens were characterized by SEM-EDS (Scanning Electron Microscopy coupled with Energy Dispersion System), XRD (XRay Diffraction), XRF (X-Ray Fluorescence), FTIR (Fourier Transform Infrared Spectroscopy) and color measurements. It was also made a microscopic and macroscopic analysis of the stone surface along with the tests of short and long term capillary absorption. A series of test trials were conducted in order to understand which concentrations and contact times best suits to this purpose, to confirm what had been written to date in the literature. We sought to develop new methods of treatment application, skipping the usual methods of applying chemical treatments on stone substrates, with the use of cellulose poultice, resorting to the agar, a gel already used in many other areas, being something new in this area, which possesses great applicability in the field of conservation of stone materials. After the application of the best methodology for cleaning, specimens were characterized again in order to understand which treatment was more effective and less harmful, both for the operator and the stone material. Very briefly conclusions were that for a very intense and deep penetration into the stone, a solution of 3.5% of SDT buffered with ammonium carbonate to pH around 7 applied with agar support would be indicated. For rust stains in its initial state, the use of Ammonium citrate at a concentration of 5% buffered with ammonium to pH 7 could be applied more than once until satisfactory results appear.

Soft biomaterials as a substitute for the Nucleus Pulposus

One of the largest health problems faced worldwide, when evaluated by direct (clinical) as well indirect cost (absenteeism), is the degeneration of the intervertebral disc (IVD) that leads to back pain and, potentially disability and individual´s quality of life decreasing. The intervertebral disc is a mechanical and biological complex structure, formed by a tough outer layer of fibrocartilage called Annulus Fibrosus (AF),which surrounds a soft, elastic and gelatinous core called Nucleus Pulposus (NP). These two structures are completed by two upper and lower encasing layer called Vertebral Endplates (VEP). The degeneration of the IVD is marked by the dehydration of the Nucleus Pulposus, reducing the hydrostatic pressure inside the nucleus, resulting in a loss of capability to support compressive forces, during the active period, and to regain height during the resting period. This situation will compromise the role of shock absorber by the NP and transfers these forces to the AF. This transfer will result in cracks on the AF, deteriorating the IVD, allowing the ingrowth of vessels and nerves. This project was based on the developing a protocol to test suitable NP replacements, in hope to future assessment of discrete mechanical values and characteristics for an NP replacement. For this, Nucleus pulposus samples from goat, encapsulated Hydromed gel denominated “Raviolis” and Chitosan gels, produced via wet route using an ammonium environment, were confined compressed. Chitosan was rheologically tested and swelling capability of all the three type of materials was assessed. Results showed that the Nucleus Pulposus and “Raviolis” have similar mechanical behavior, being able to swell and “build up” hydrostatic pressure after a compression stage, while the Chitosan gel did not showed that ability. Therefore, “Raviolis” are a more suitable candidate to replace the NP than Chitosan gels. It was also observed that confined compression is the key test to perform on any possible candidate to replace the NP.