Electrochemical determination of antioxidant capacities in flavored waters by guanine and adenine biosensors

The immobilization and electro-oxidation of guanine and adenine asDNA bases on glassy carbon electrode are evaluated by square wave voltammetric analysis. The influence of electrochemical pretreatments, nature of supporting electrolyte, pH, accumulation time and composition of DNA nucleotides on the immobilization effect and the electrochemical mechanism are discussed. Trace levels of either guanine or adenine can be readily detected following short accumulation time with detection limits of 35 and 40 ngmL−1 for guanine and adenine, respectively. The biosensors of guanine and adenine were employed for the voltammetric detection of antioxidant capacity in flavored water samples. The method relies on monitoring the changes of the intrinsic anodic response of the surface-confined guanine and adenine species, resulting from its interaction with free radicals from Fenton-type reaction in absence and presence of antioxidant. Ascorbic acid was used as standard to evaluate antioxidant capacities of samples. Analytical data was compared with that of FRAP method.

DNA-based biosensor for the electrocatalytic determination of antioxidant capacity in beverages

Reactive oxygen species (ROS) are produced as a consequence of normal aerobic metabolism and are able to induce DNA oxidative damage. At the cellular level, the evaluation of the protective effect of antioxidants can be achieved by examining the integrity of the DNA nucleobases using electrochemical techniques. Herein, the use of an adenine-rich oligonucleotide (dA21) adsorbed on carbon paste electrodes for the assessment of the antioxidant capacity is proposed. The method was based on the partial damage of a DNA layer adsorbed on the electrode surface by OH• radicals generated by Fenton reaction and the subsequent electrochemical oxidation of the intact adenine bases to generate an oxidation product that was able to catalyze the oxidation of NADH. The presence of antioxidant compounds scavenged hydroxyl radicals leaving more adenines unoxidized, and thus, increasing the electrocatalytic current of NADHmeasured by differential pulse voltammetry (DPV). Using ascorbic acid (AA) as a model antioxidant species, the detection of as low as 50nMof AA in aqueous solution was possible. The protection efficiency was evaluated for several antioxidant compounds. The biosensor was applied to the determination of the total antioxidant capacity (TAC) in beverages.

Determination of polyphenols in wines by reaction with 4-aminoantipyrine and photometric flow-injection analysis

A new flow-injection analytical procedure is proposed for the determination of the total amount of polyphenols in wines; the method is based on the formation of a colored complex between 4-aminoantipyrine and phenols, in the presence of an oxidizing reagent. The oxidizing agents hexacyanoferrate(III), peroxodisulfate, and tetroxoiodate(VII) were tested. Batch trials were first performed to select appropriate oxidizing agents, pH, and concentration ratios of reagents, on the basis of their effect on the stability of the colored complex. Conditions selected as a result of these trials were implemented in a flow-injection analytical system in which the influence of injection volume, flow rate, and reaction- coil length, was evaluated. Under the optimum conditions the total amount of polyphenols, expressed as gallic acid, could be determined within a concentration range of 36 to 544 mg L–1, and with a sensitivity of 344 L mol–1 cm–1 and an RSD <1.1%. The reproducibility of analytical readings was indicative of standard deviations <2%. Interference from sugars, tartaric acid, ascorbic acid, methanol, ammonium sulfate, and potassium chloride was negligible. The proposed system was applied to the determination of total polyphenols in red wines, and enabled analysis of approximately 55 samples h–1. Results were usually precise and accurate; the RSD was <3.9% and relative errors, by the Folin–Ciocalteu method, <5.1%.

Electrochemical DNA-sensor for evaluation of total antioxidant capacity of flavours and flavoured waters using superoxide radical damage

In this paper, a biosensor based on a glassy carbon electrode (GCE) was used for the evaluation of the total antioxidant capacity (TAC) of flavours and flavoured waters. This biosensor was constructed by immobilising purine bases, guanine and adenine, on a GCE. Square wave voltammetry (SWV) was selected for the development of this methodology. Damage caused by the reactive oxygen species (ROS), superoxide radical (O2·−), generated by the xanthine/xanthine oxidase (XOD) system on the DNA-biosensor was evaluated. DNA-biosensor encountered with oxidative lesion when it was in contact with the O2·−. There was less oxidative damage when reactive antioxidants were added. The antioxidants used in this work were ascorbic acid, gallic acid, caffeic acid, coumaric acid and resveratrol. These antioxidants are capable of scavenging the superoxide radical and therefore protect the purine bases immobilized on the GCE surface. The results demonstrated that the DNA-based biosensor is suitable for the rapid assess of TAC in beverages.

Electrocatalytic evaluation of DNA damage by superoxide radical for antioxidant capacity assessment

The integrity of DNA purine bases was herein used to evaluate the antioxidant capacity. Unlike other DNA-based antioxidant sensors reported so far, the damaging agent chosen was the O 2 radical enzymatically generated by the xanthine/xanthine oxidase system. An adenine-rich oligonucleotide was adsorbed on carbon paste electrodes and subjected to radical damage in the presence/absence of several antioxidant compounds. As a result, partial damage on DNA was observed. A minor product of the radical oxidation was identified by cyclic voltammetry as a diimine adenine derivative also formed during the electrochemical oxidation of adenine/guanine bases. The protective efficiency of several antioxidant compounds was evaluated after electrochemical oxidation of the remaining unoxidized adenine bases, by measuring the electrocatalytic current of NADH mediated by the adsorbed catalyst species generated. A comparison between O 2 and OH radicals as a source of DNA lesions and the scavenging efficiency of various antioxidant compounds against both of them is discussed. Finally, the antioxidant capacity of beverages was evaluated and compared with the results obtained with an optical method.

Evaluation of the total antioxidant capacity of flavored water and electrochemical purine damage by sulfate radicals using a purine-based sensor

In this study, a method for the electrochemical quantification of the total antioxidant capacity (TAC) in beverages was developed. The method is based on the oxidative damage to the purine bases, adenine or guanine, that are immobilized on a glassy carbon electrode (GCE) surface. The oxidative lesions on the DNA bases were promoted by the sulfate radical generated by the persulfate/iron(II) system. The presence of antioxidants on the reactive system promoted the protection of the DNA bases immobilized on the GCE by scavenging the sulfate radical. Square-wave voltammetry (SWV) was the electrochemical technique used to perform this study. The efficiencies of five antioxidants (ascorbic acid, gallic acid, caffeic acid, coumaric acid and resveratrol) in scavenging the sulfate radical and, therefore, their ability to protect the purine bases immobilized on the GCE were investigated. These results demonstrated that the purine-based biosensor is suitable for the rapid assessment of the TAC in flavors and flavored water.

Molecularly-imprinted materials for potentiometric transduction: application to the antibiotic enrofloxacin

Enrofloxacin (ENR) is an antimicrobial used both in humans and in food producing species. Its control is required in farmed species and their surroundings in order to reduce the prevalence of antibiotic resistant bacteria. Thus, a new biomimetic sensor enrofloxacin is presented. An artificial host was imprinted in specific polymers. These were dispersed in 2-nitrophenyloctyl ether and entrapped in a poly(vinyl chloride) matrix. The potentiometric sensors exhibited a near-Nernstian response. Slopes expressing mVΔlog([ENR]/M) varied within 48–63. The detection limits ranged from 0.28 to 1.01 µg mL 1. Sensors were independent from the pH of test solutions within 4–7. Good selectivity was observed toward potassium, calcium, barium, magnesium, glycine, ascorbic acid, creatinine, norfloxacin, ciprofloxacin, and tetracycline. In flowing media, the biomimetic sensors presented good reproducibility (RSD of ±0.7%), fast response, good sensitivity (47 mV/Dlog([ENR]/ M), wide linear range (1.0×10-5–1.0×10-3 M), low detection limit (0.9 µg mL-1), and a stable baseline for a 5×10-2 M acetate buffer (pH 4.7) carrier. The sensors were used to analyze fish samples. The method offered the advantages of simplicity, accuracy, and automation feasibility. The sensing membrane may contribute to the development of small devices allowing in vivo measurements of enrofloxacin or parent-drugs.

Control and comparison of the antioxidant capacity of beers

The purpose of the present work is to determine the antioxidant capacity (AC) of 27 commercial beers. The AC indicates the degree of protection of a certain organism against oxidative damage provoked by reactive oxygen and nitrogen species. Assays were carried out by the following methods: (i) total radical trapping antioxidant parameter (TRAP); (ii) trolox equivalent antioxidant capacity (TEAC); (iii) trolox equivalent antioxidant capacity (DPPH); (iv) ferric-ion reducing antioxidant parameter (FRAP); (v) cupric reducing antioxidant capacity (CUPRAC); (vi) oxygen radical absorbance capacity (ORAC). Ascorbic acid (AA), gallic acid (GA) and trolox (TR) were used as standards. All beers showed antioxidant power, but a wide range of ACs was observed. The effect of several factors upon these differences was studied. Statistical differences were found between ACs of beers of different colours. ORAC method provided always higher experimental ACs, of significant statistical differences to other assays.

Protein-Polysaccharides of trametes versocolor: production and biological activities

Extracellular-(E-PPS) and intracellular-protein-polysaccharides (I-PPS) complexes were produced by Trametes versicolor in submerged cultures with different carbon sources. The highest extracellular-(EPS) and intracellular-polysaccharide (IPS) concentration in the complexes was obtained with tomato pomace culture. DPPH radical scavenging for E-PPS and I-PPS produced by liter of culture was equivallent to 2.115 +/- A 0.227 and 1.374 +/- A 0.364 g of ascorbic acid, respectively. These complexes showed a protector effect in the oxidation of erythrocyte membranes and had ability to inhibit the hemolysis and methemoglobin synthesis in stressed erythrocytes. These results suggest that extracellular- and intracellular- polysaccharides produced are important bioactive compounds with medicinal potential.

Effect of peel and seed removal on the nutritional value and antioxidant activity of tomato (Lycopersicon esculentum L.) fruits

The effect of peel and seed removal, two commonly practiced procedures either at home or by the processing industry, on the physicochemical properties, bioactive compounds contents and antioxidant capacity of tomato fruits of four typical Portuguese cultivars (cereja, chucha, rama and redondo) were appraised. Both procedures caused significant nutritional and antioxidant activity losses in fruits of every cultivar. In general, peeling was more detrimental, since it caused a higher decrease in lycopene, bcarotene, ascorbic acid and phenolics contents (averages of 71%, 50%, 14%, and 32%, respectively) and significantly lowered the antioxidant capacity of the fruits (8% and 10%, using DPPH. and b-carotene linoleate model assays, correspondingly). Although seeds removal favored the increase of both color and sweetness, some bioactive compounds (11% of carotenoids and 24% of phenolics) as well as antioxidant capacity (5%) were loss. The studied cultivars were differently influenced by these procedures. The fruits most affected by peeling were those from redondo cultivar (-66% lycopene, -44% b-carotene, -26% ascorbic acid and -38% phenolics). Seeds removal, in turn, was more injurious for cereja tomatoes (-10% lycopene, -38% b-carotene, -25% ascorbic acid and -63% phenolics). Comparatively with the remaining ones, the rama fruits were less affected by the trimming procedures.

Sensitive bi-enzymatic biosensor based on polyphenoloxidases–gold nanoparticles–chitosan hybrid film–graphene doped carbon paste electrode for carbamates detection

A bi-enzymatic biosensor (LACC–TYR–AuNPs–CS/GPE) for carbamates was prepared in a single step by electrodeposition of a hybrid film onto a graphene doped carbon paste electrode (GPE). Graphene and the gold nanoparticles (AuNPs) were morphologically characterized by transmission electron microscopy, X-ray photoelectron spectroscopy, dynamic light scattering and laser Doppler velocimetry. The electrodeposited hybrid film was composed of laccase (LACC), tyrosinase (TYR) and AuNPs entrapped in a chitosan (CS) polymeric matrix. Experimental parameters, namely graphene redox state, AuNPs:CS ratio, enzymes concentration, pH and inhibition time were evaluated. LACC–TYR–AuNPs–CS/GPE exhibited an improved Michaelis–Menten kinetic constant (26.9 ± 0.5 M) when compared with LACC–AuNPs–CS/GPE (37.8 ± 0.2 M) and TYR–AuNPs–CS/GPE (52.3 ± 0.4 M). Using 4-aminophenol as substrate at pH 5.5, the device presented wide linear ranges, low detection limits (1.68×10− 9 ± 1.18×10− 10 – 2.15×10− 7 ± 3.41×10− 9 M), high accuracy, sensitivity (1.13×106 ± 8.11×104 – 2.19×108 ± 2.51×107 %inhibition M− 1), repeatability (1.2–5.8% RSD), reproducibility (3.2–6.5% RSD) and stability (ca. twenty days) to determine carbaryl, formetanate hydrochloride, propoxur and ziram in citrus fruits based on their inhibitory capacity on the polyphenoloxidases activity. Recoveries at two fortified levels ranged from 93.8 ± 0.3% (lemon) to 97.8 ± 0.3% (orange). Glucose, citric acid and ascorbic acid do not interfere significantly in the electroanalysis. The proposed electroanalytical procedure can be a promising tool for food safety control.

Estudo eletroquímico do dano oxidativo promovido por RNOS e PAH e o efeito protetor de antioxidantes no ADN

No dia-a-dia, os organismos vivos estão sujeitos a vários tipos de agressões de origem endógena e exógena. A produção endógena exagerada de agentes oxidantes que ocorre nos processos metabólicos dos seres vivos está intimamente associada ao aparecimento e desenvolvimento de várias patologias. Por outro lado, e devido às atividades antropogénicas, muitos agentes oxidantes de origem ambiental e alimentar entram por via exógena no organismo dos seres vivos provocando igualmente danos a nível celular. De modo a protegerem-se dos efeitos pejorativos provocados por estes compostos, os organismos vivos desenvolveram mecanismos complexos de defesa antioxidante. Este trabalho consistiu no estudo eletroquímico do dano oxidativo induzido por agentes oxidantes (PAH (hidrocarbonetos aromáticos policíclicos), H2O2, NO• e HClO) e do efeito protetor, ao dano oxidativo, promovido por antioxidantes no material baseado no ADN recorrendo à utilização de um biossensor de bases púricas, adenina-EPC (elétrodo pasta de carbono) e dA20-EPC, utilizando a voltametria de onda quadrada (VOQ) como técnica de deteção. A aplicação da eletroquímica apresenta várias vantagens para a quantificação da capacidade antioxidante total (CAT) pois, permite a redução da quantidade de reagentes e amostra em análise, elimina a etapa de remoção de cor (a cor é um interferente nos métodos óticos) e não requer equipamentos dispendiosos. Foram seguidas diferentes abordagens para a construção dos biossensores. A primeira consistiu na construção de um adenina-EPC em três etapas: i) condicionamento do EPC, ii) eletrodeposição da adenina no EPC e iii) leitura do sinal eletroquímico. Assim, foram otimizados diversos parâmetros: concentração de adenina (150,0 mg/L), potencial de condicionamento (Ec) (+ 1,80 V), potencial de deposição (Ed) (+ 0,40 V), tempo de condicionamento (tc) (180 s) e tempo de deposição (td) (240 s). Foi aplicado o adenina-EPC no estudo do dano oxidativo provocado por PAH (benzo (g,h,i) perileno) e constatou-se que era necessário transformar o benzo (g,h,i) perileno num radical para se possível observar danos oxidativos induzidos no biossensor. A nova estratégia consistiu na construção de um dA20-EPC, através da adsorção física de uma gota de dA20 na superfície do EPC, com posterior secagem e leitura do sinal eletroquímico. Neste procedimento foi otimizada a concentração de dA20 (100,0 mg/L). O dano oxidativo provocado pelo H2O2, NO• e HClO foi estudado sobre o dA20-EPC e verificou-se que os três agentes oxidantes induziam dano oxidativo no dA20-EPC. Confirmou-se a capacidade do ácido ascórbico (AA) em proteger o dA20-EPC do dano oxidativo induzido por H2O2 e NO•. O biossensor desenvolvido (dA20-EPC) foi aplicado na avaliação da CAT de diferentes amostras reais (café, sumo de laranja e água aromatizada de laranja) usando-se como agentes oxidantes o H2O2 e NO•. Todas as amostras analisadas apresentaram ter capacidade antioxidante. Quando se utilizou o dA20-EPC na presença de H2O2, verificou-se que as amostras de café apresentam valores mais elevados de CAT (1130-1488 mg AAE/L) do que as amostras de bebidas (110 mg AAE/L em água aromatizada e 775 mg AAE/L em sumo). Os valores de CAT obtidos para amostras de sumo e água aromatizada na presença de NO• indicam que a amostra de sumo possui maior teor de CAT (871 mg AAE/L) conforme era esperado, do que a amostra de água aromatizada (172 mg AAE/L). Na presença de HClO, o valor de CAT mais elevado pertence a uma amostra de sumo (513 mg AAE/L) mas, o valor de CAT da amostra de sumo natural é muito mais baixa do que o esperado (17 mg AAE/L). Foram estudados outros antioxidantes para além do AA (ácido cumárico, ácido gálico e ácido cafeico), e constatou-se que cada um deles promove proteção ao dA20-EPC na presença de cada um dos diferentes contaminantes (H2O2, NO• e HClO).

Desenvolvimento e aplicação de um biossensor para monitorização de creatinina, um biomarcador associado a doenças emergentes

Este trabalho descreve o desenvolvimento de um material sensor para creatinina por impressão molecular em estrutura polimérica (MIP) e a sua aplicação no desenvolvimento de um dispositivo de natureza potenciométrica para a determinação da molécula alvo em fluidos biológicos. A creatinina é um dos biomarcadores mais utilizados no acompanhamento da doença renal, já que é um bom indicador da taxa de filtração glomerular (TFG). Os materiais biomiméticos desenhados para interação com a creatinina foram obtidos por polimerização radicalar, recorrendo a monómeros de ácido metacríclico ou de vinilpiridina e a um agente de reticulação apropriado. De modo a aferir o efeito da impressão da creatinina na resposta dos materiais MIP à sua presença, foram também preparados e avaliados materiais de controlo, obtidos sem impressão molecular (NIP). O controlo da constituição química destes materiais, incluindo a extração da molécula impressa, foi realizado por Espectroscopia de Raman e de Infravermelho com Transformada de Fourrier. A afinidade de ligação entre estes materiais e a creatinina foi também avaliada com base em estudos cinéticos. Todos os materiais descritos foram integrados em membranas selectivas de elétrodos seletivos de ião, preparadas sem ou com aditivo iónico lipófilo, de carga negativa ou positiva. A avaliação das características gerais de funcionamento destes elétrodos, em meios de composição e pH diferentes, indicaram que as membranas com materiais impressos e aditivo aniónico eram as únicas com utilidade analítica. Os melhores resultados foram obtidos em solução tampão Piperazine-N,N′-bis(2- ethanesulfonic acid), PIPES, de pH 2,8, condição que permitiu obter uma resposta quasi-Nernstiana, a partir de 1,6×10-5 mol L-1. Estes elétrodos demonstraram ainda uma boa selectividade ao apresentaram uma resposta preferencial para a creatinina quando na presença de ureia, carnitina, glucose, ácido ascórbico, albumina, cloreto de cálcio, cloreto de potássio, cloreto de sódio e sulfato de magnésio. Os elétrodos foram ainda aplicados com sucesso na análise de amostras sintéticas de urina, quando os materiais sensores eram baseados em ácido metacrilico, e soro, quando os materiais sensores utilizados eram baseados em vinilpiridina.

Graphene-based biomimetic materials targeting urine metabolite as potential cancer biomarker: Application over different conductive materials for potentiometric transduction

This work presents a novel surface Smart Polymer Antibody Material (SPAM) for Carnitine (CRT, a potential biomarker of ovarian cancer), tested for the first time as ionophore in potentiometric electrodes of unconventional configuration. The SPAM material consisted of a 3D polymeric network created by surface imprinting on graphene layers. The polymer was obtained by radical polymerization of (vinylbenzyl) trimethylammonium chloride and 4-styrenesulfonic acid (signaling the binding sites), and vinyl pivalate and ethylene glycol dimethacrylate (surroundings). Non-imprinted material (NIM) was prepared as control, by excluding the template from the procedure. These materials were then used to produce several plasticized PVC membranes, testing the relevance of including the SPAM as ionophore, and the need for a charged lipophilic additive. The membranes were casted over solid conductive supports of graphite or ITO/FTO. The effect of pH upon the potentiometric response was evaluated for different pHs (2-9) with different buffer compositions. Overall, the best performance was achieved for membranes with SPAM ionophore, having a cationic lipophilic additive and tested in HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, pH 5.1. Better slopes were achieved when the membrane was casted on conductive glass (-57.4 mV/decade), while the best detection limits were obtained for graphite-based conductive supports (3.6 × 10−5mol/L). Good selectivity was observed against BSA, ascorbic acid, glucose, creatinine and urea, tested for concentrations up to their normal physiologic levels in urine. The application of the devices to the analysis of spiked samples showed recoveries ranging from 91% (± 6.8%) to 118% (± 11.2%). Overall, the combination of the SPAM sensory material with a suitable selective membrane composition and electrode design has lead to a promising tool for point-of-care applications.

Assessing and comparing the total antioxidant capacity of commercial beverages: application to beers, wines, waters and soft drinks using TRAP, TEAC and FRAP methods.

This work measures and tries to compare the Antioxidant Capacity (AC) of 50 commercial beverages of different kinds: 6 wines, 12 beers, 18 soft drinks and 14 flavoured waters. Because there is no reference procedure established for this purpose, three different optical methods were used to analyse these samples: Total Radical trapping Antioxidant Parameter (TRAP), Trolox Equivalent Antioxidant Capacity (TEAC) and Ferric ion Reducing Antioxidant Parameter (FRAP). These methods differ on the chemical background and nature of redox system. The TRAP method involves the transfer of hydrogen atoms while TEAC and FRAP involves electron transfer reactions. The AC was also assessed against three antioxidants of reference, Ascorbic acid (AA), Gallic acid (GA) and 6-hydroxy-2,5,7,8-tetramethyl- 2-carboxylic acid (Trolox). The results obtained were analyzed statistically. Anova one-way tests were applied to all results and suggested that methods and standards exhibited significant statistical differences. The possible effect of sample features in the AC, such as gas, flavours, food colouring, sweeteners, acidity regulators, preservatives, stabilizers, vitamins, juice percentage, alcohol percentage, antioxidants and the colour was also investigated. The AC levels seemed to change with brand, kind of antioxidants added, and kind of flavour, depending on the sample. In general, higher ACs were obtained for FRAP as method, and beer for kind of sample, and the standard expressing the smaller AC values was GA.