Peróxido de hidrogênio: importância e determinação

A brief discussion about the hydrogen peroxide importance and its determination is presented. It was emphasized some consideration of the H2O2 as reagent (separated or combined), uses and methods of analysis (techniques, detection limits, linear response intervals, sensor specifications). Moreover, it was presented several applications, such as in environmental, pharmaceutical, medicine and food samples.

Utilização de técnicas eletroanalíticas na determinação de pesticidas em alimentos

The aim of this work is to discuss selected applications of electroanalytical techniques for the detection of pesticides in foods and beverages, published in the last ten years. The applications involved different working electrodes for the electroanalytical determination of pesticides, namely amperometric biosensors, cholinesterase-based biosensors, polymer-modified electrodes, ultramicroelectrodes and hanging mercury drop electrodes. They were used for several voltammetric and amperometric techniques in different analytical procedures for the detection and quantification of different classes of pesticides in different food matrices.

Filmes de nanodiamantes para aplicações em sistemas eletroquímicos e tecnologia aeroespacial

The goal of this work is to show the use of undoped nanodiamond films as a new material for electrochemical and aerospace applications. Correlation between the applications and physico-chemical features of nano and conventional CVD polycrystalline diamond films are presented. An important and innovative application of these nanodiamonds is organic electrosynthesis, including pharmaceutical and water disinfection products, as well as electroanalytical applications, for example, development of biosensors for detection of glucose, glutamate and dopamine. In aeronautics and space developments, these nanodiamonds could be used as electrodes in rechargable batteries and in tribological investigations.

Construção e aplicação de biossensores usando diferentes procedimentos de imobilização da peroxidase de vegetal em matriz de quitosana

Biosensors were developed by immobilization of gilo (Solanum gilo) enzymatic extract on chitosan biopolymers using three different procedures: glutaraldehyde, carbodiimide/glutaraldehyde and epichlorohydrin/glutaraldehyde. The best biosensor performance was obtained after the immobilization of peroxidase on chitosan with epichlorohydrin/glutaraldehyde. Linear analytical curves for hydroquinone concentrations from 2.5x10-4 to 4.5x10-3 mol L-1 with a detection limit of 2.0x10-6 mol L-1 and recovery of hydroquinone ranging from 95.1 to 105% were obtained. The relative standard deviation was < 1.0 % for a solution of 3.0x10-4 mol L-1 hydroquinone and 2.0x10-3 mol L-1 hydrogen peroxide in 0.1 mol L-1 phosphate buffer solution at pH 7.0 (n=8). The lifetime of this biosensor was 6 months (at least 300 determinations).

Biossensor enzimático para detecção de fungicidas ditiocarbamatos: estudo cinético da enzima aldeído desidrogenase e otimização do biossensor

Initially, all major factors that affect the rate of the AldH-catalyzed reaction (enzyme concentration, substrate concentration, temperature and pH) were investigated. Optimal activity was observed between pH values of 7.5 and 9.5 in the temperature range of 25 to 50 ºC. Kinetic parameters, such as Km (2.92 µmol L-1) and Vmax (1.33 10-2 µmol min-1) demonstrate a strong enzyme-substrate affinity. The sensors were based on screen-printed electrodes modified with the Meldola Blue-Reinecke salt (MBRS) combination. Operational conditions (NAD+ and substrate contents, enzyme loading and response time) were optimized. Also, two enzyme immobilization procedures were tested: entrapment in poly(vinyl alcohol) bearing styrylpyridinium groups (PVA-SbQ) and crosslinking with glutaraldehyde. Chronoamperometry was employed to observe the biosensor responses during enzymatic hydrolysis of propionaldehyde and also to construct inhibition curves with maneb and zineb fungicides. Best results were found with the following conditions: [NAD+] = 0.25 mmol L-1; [propionaldehyde] = 80 µmol L-1; enzyme loading = 0.8 U per electrode; response time = 10 min, and inhibition time = 10 min. Current intensities around 103 ± 13 nA with the sensors and good stability was obtained for both immobilization procedures. Detection limits, calculated using 10% inhibition were 31.5 µg L-1 and 35 µg L-1 for maneb and zineb, respectively. Results obtained with other MBRS-modified electrodes consisting of mono and bi-enzymic sensors were compared. The ability to catalyze NADH oxidation by MB was also highlighted.

Biossensores baseados no processo de inibição enzimática

Fast, selective, reproducible and reliable detections have been carried out by using enzymatic biosensors in several areas. The enzymatic biosensors based on the inhibition represent an important role in analytical chemistry. Enzymes like cholinesterases, peroxidases, tyrosinases, etc. have been immobilized on electrochemical and optical transducers and the enzymatic activity decreasing in the presence of the inhibitor is related with its concentrations. This article presents a review on the enzymes used on the construction of these sensors, emphasizing the respective applications.

Imobilização da lacase em micropartículas de quitosana obtidas por spray drying e usadas na construção de biossensores

Biosensors based on laccase immobilized on microparticles of chitosan crosslinked with tripolyphosphate (biosensor I) and glyoxal (biosensor II) obtained by spray drying for the determinations of rutin in pharmaceutical formulations were developed. Under optimized operational conditions (pH 4.0, frequency of 30 Hz, pulse amplitude of 40 mV and scan increment of 2.0 mV) two analytical curves were obtained for both biosensors showing a detection limit of 6.2x10-8 mol L-1 for biosensor (I) and 2.0x10-8 mol L-1 for biosensor (II). The recovery of rutin from pharmaceutical sample ranged from 90.7 to 105.0% and the lifetime of these biosensors were 4 months (at least 400 determinations).

Incorporação de líquidos iônicos e nanopartículas metálicas na construção de sensores eletroquímicos

The most relevant advances on analytical applications of ionic liquids (IL) as binder in the construction of electrochemical sensors and biosensors based on carbon paste are presented. This new class of solvents - the IL - has received great attention in electroanalytical researches due to the excellent physical and chemical properties of these materials, such as high conductivity, low toxicity, good stability, large electrochemical window and catalytic ability. Recently, the interest in electrodes modified with IL, especially when combined with metallic nanoparticles, has increased expressively due to improve the sensitivity and others advantages discussed in this review.

BIOSSENSOR ELETROQUÍMICO BASEADO NA ENZIMA TIROSINASE PARA A DETERMINAÇÃO DE FENOL EM EFLUENTES

AbstractThis work describes the development of a biosensor based on the tyrosinase enzyme (Tyr) for the determination of phenol (PHEN) in laboratory effluent samples derived from ammoniacal nitrogen analysis of the water samples from the Muquém dam in the city of Cariús, CE, using square-wave voltammetry (SWV). The electrode modification consisted of the immobilization of gold nanoparticles, multi-walled carbon nanotubes, cobalt phthalocyanine, and Tyr on a glassy carbon electrode. The electrolyte, pH, enzyme quantity, and voltammetric parameters were optimized to detect PHEN. The analytical curves presented a linear range from 4.97 × 10-6 mol L-1 to 6.10 × 10-5 mol L-1, and the detection limit (DL) and quantitation limit (QL) values were 4.81 × 10-6 mol L-1 and 4.97 × 10-6mol L-1, respectively. The repetition of measurements with the same biosensor and repetition for three other prepared biosensors exhibited a relative standard deviation (RSD) of 5.50 and 1.75%, respectively. The percentage recovery of PHEN in effluent samples varied from 86.40 to 105.04%. The stability of the biosensor was evaluated (at 21 days) with satisfactory results, showing 97.86% of the initial response. Moreover, the DL and recovery percentages agreed with the established values from CONAMA and ABNT, respectively. Thus, the electrode configuration developed seems a promising tool in the detection and quantification of PHEN in complex samples.

Determination of piglets’ rectal temperature and respiratory rate through skin surface temperature under climatic chamber conditions

ABSTRACT In animal farming, an automatic and precise control of environmental conditions needs information from variables derived from the animals themselves, i.e. they act as biosensors. Rectal temperature (RT) and respiratory rate (RR) are good indicators of thermoregulation in pigs. Since there is a growing concern on animal welfare, the search for alternatives to measure RT has become even more necessary. This research aimed to identify the most adequate body surface areas, on nursery-phase pigs, to take temperature measurements that best represent the correlation of RT and RR. The main experiment was carried out in a climate chamber with five 30-day-old littermate female Landrace x Large White piglets. Temperature conditions inside chamber were varied from 14 °C up to 35.5 °C. The measurements were taken each 30 minutes, over six different skin regions, using a temperature data logger Thermochron iButton® - DS1921G (Tb) and an infrared thermometer (Ti). As shown by the results, the tympanic region is the best one for RT and RR monitoring using an infrared thermometer (TiF). In contrast, when using temperature sensors, the ear (TbE) is preferred to be used for RT predictions and the loin region (TbC) for RR.

Porous silicon optical filters in gas sensing applications

In this thesis, the gas sensing properties of porous silicon-based thin-film optical filters are explored. The effects of surface chemistry on the adsorption and desorption of various gases are studied in detail. Special emphasis is placed on investigating thermal carbonization as a stabilization method for optical sensing applications. Moreover, the possibility of utilizing the increased electrical conductivity of thermally carbonized porous silicon for implementing a multiparametric gas sensor, which would enable simultaneous monitoring of electrical and optical parameters, is investigated. In addition, different porous silicon-based optical filter-structures are prepared, and their properties in sensing applications are evaluated and compared. First and foremost, thermal carbonization is established as a viable method to stabilize porous silicon optical filters for chemical sensing applications. Furthermore, a multiparametric sensor, which can be used for increasing selectivity in gas sensing, is also demonstrated. Methods to improve spectral quality in multistopband mesoporous silicon rugate filters are studied, and structural effects to gas sorption kinetics are evaluated. Finally, the stability of thermally carbonized optical filters in basic environments is found to be superior in comparison to other surface chemistries currently available for porous silicon. The results presented in this thesis are of particular interest for developing novel reliable sensing systems based on porous silicon, e.g., label-free optical biosensors.

Regioselective modification of galactose-containing polysaccharides in aqueous media

Biorefining is defined as sustainable conversion of biomass into marketable products and energy. Forests cover almost one third of earth’s land area, and account for approximately 40% of the total annual biomass production. In forest biorefining, the wood components are, in addition to the traditional paper and board products, converted into chemicals and biofuels. The major components in wood are cellulose, hemicelluloses, and lignin. The main hemicellulose in softwoods, which are of interest especially for the Nordic forest industry, is O-acetyl galactoglucomannan (GGM). GGM can be isolated in industrial scale from the waste waters of the mechanical pulping process, but is not yet today industrially utilized. In order to attain desired properties of GGM for specific end-uses, chemical and enzymatic modifications can be performed. Regioselective modifications of GGM, and other galactose-containing polysaccharides were done by oxidations, and by combining oxidations with subsequent derivatizations of the formed carbonyl or carboxyl groups. Two different pathways were investigated: activation of the C-6 positions in different sugar units by TEMPO-mediated oxidation, and activation of C-6 position in only galactose-units by oxidation catalyzed by the enzyme galactose oxidase. The activated sites were further selectively derivatized; TEMPO-oxidized GGM by a carbodiimide-mediated reaction forming amides, and GO-oxidized GGM by indium-mediated allylation introducing double or triple bonds to the molecule. In order to better understand the reaction, and to develop a MALDI-TOF-MS method for characterization of regioselectively allylated GGM, α-D-galactopyranoside and raffinose were used as model compounds. All reactions were done in aqueous media. To investigate the applicability of the modified polysaccharides for, e.g., cellulose surface functionalization, their sorption onto pulp fibres was studied. Carboxylation affects the sorption tendency significantly; a higher degree of oxidation leads to lower sorption. By controlling the degree of oxidation of the polysaccharides and the ionic strength of the sorption media, high degrees of sorption of carboxylated polysaccharides onto cellulose could, however, be obtained. Anionic polysaccharides were used as templates during laccase-catalyzed polymerization of aniline, offering a green, chemo-enzymatic route for synthesis of conducting polyaniline (PANI) composite materials. Different polysaccharide templates, such as, native GGM, TEMPO-oxidized GGM, naturally anionic κ-carrageenan, and nanofibrillated cellulose produced by TEMPO-oxidation, were assessed. The conductivity of the synthesized polysaccharide/PANI biocomposites varies depending on the polysaccharide template; κ-CGN, the anionic polysaccharide with the lowest pKa value, produces the polysaccharide/PANI biocomposites with the highest conductivity. The presented derivatization, sorption, and polymerization procedures open new application windows for polysaccharides, such as spruce GGM. The modified polysaccharides and the conducting biocomposites produced provide potential applications in biosensors, electronic devices, and tissue engineering.

Targeting oncogenic signaling proteins : new insights using a FRET-based chemical biology approach

Cancer affects more than 20 million people each year and this rate is increasing globally. The Ras/MAPK-pathway is one of the best-studied cancer signaling pathways. Ras proteins are mutated in almost 20% of all human cancers and despite numerous efforts, no effective therapy that specifically targets Ras is available to date. It is now well established that Ras proteins laterally segregate on the plasma membrane into transient nanoscale signaling complexes called nanoclusters. These Ras nanoclusters are essential for the high-fidelity signal transmission. Disruption of nanoclustering leads to reduction in Ras activity and signaling, therefore targeting nanoclusters opens up important new therapeutic possibilities in cancer. This work describes three different studies exploring the idea of membrane protein nanoclusters as novel anti-cancer drug targets. It is focused on the design and implementation of a simple, cell-based Förster Resonance Energy Transfer (FRET)-biosensor screening platform to identify compounds that affect Ras membrane organization and nanoclustering. Chemical libraries from different sources were tested and a number of potential hit molecules were validated on full-length oncogenic proteins using a combination of imaging, biochemical and transformation assays. In the first study, a small chemical library was screened using H-ras derived FRET-biosensors. Surprisingly from this screen, commonly used protein synthesis inhibitors (PSIs) were found to specifically increase H-ras nanoclustering and downstream signalling in a H-ras dependent manner. Using a representative PSI, increase in H-ras activity was shown to induce cancer stem cell (CSC)-enriched mammosphere formation and tumor growth of breast cancer cells. Moreover, PSIs do not increase K-ras nanoclustering, making this screening approach suitable for identifying Ras isoform-specific inhibitors. In the second study, a nanoncluster-directed screen using both H- and K-ras derived FRET biosensors identified CSC inhibitor salinomycin to specifically inhibit K-ras nanocluster organization and downstream signaling. A K-ras nanoclusteringassociated gene signature was established that predicts the drug sensitivity of cancer cells to CSC inhibitors. Interestingly, almost 8% of patient tumor samples in the The Cancer Genome Atlas (TCGA) database had the above gene signature and were associated with a significantly higher mortality. From this mechanistic insight, an additional microbial metabolite screen on H- and K-ras biosensors identified ophiobolin A and conglobatin A to specifically affect K-ras nanoclustering and to act as potential breast CSC inhibitors. In the third study, the Ras FRET-biosensor principle was used to investigate membrane anchorage and nanoclustering of myristoylated proteins such as heterotrimeric G-proteins, Yes- and Src-kinases. Furthermore, Yes-biosensor was validated to be a suitable platform for performing chemical and genetic screens to identify myristoylation inhibitors. The results of this thesis demonstrate the potential of the Ras-derived FRETbiosensor platform to differentiate and identify Ras-isoform specfic inhibitors. The results also highlight that most of the inhibitors identified predominantly perturb Ras subcellular distribution and membrane organization through some novel and yet unknown mechanisms. The results give new insights into the role of Ras nanoclusters as promising new molecular targets in cancer and in stem cells.

Fluorometric monitoring of the properties of proteins entrapped in sol-gel derived matrices

The successful development of stable biosensors incorporating entrapped proteins suffers from poor understanding of the properties of the entrapped biomolecules. This thesis reports on the use of fluorescence spectroscopy to investigate the properties of proteins entrapped in sol-gel processed silicate materials. Two different single tryptophan (Trp) proteins were investigated in this thesis, the Ca2 + binding protein cod III parvalbumin (C3P) and the salicylate binding protein human serum albumin (HSA). Furthermore, the reactive single cysteine (Cys) residue within C3P and HSA were labelled with the probes iodoacetoxynitrobenzoxadiazole (C3P) and acrylodan (C3P and HSA) to further examine the structure, stability and function of the free and entrapped proteins. The results show that both C3P and HSA can be successfully entrapped into sol-gelderived matrices with retention of function and conformational flexibility.

Développement d’un microréacteur à base d’enzyme microencapsulée en vue d’un couplage en ligne à un système d’électrophorèse capillaire

Réalisé en codirection avec Karen C. Waldron et Dominic Rochefort.