The sol-gel technique has drawn considerable attention in the area of biomolecules immobilization by virtue of its intrinsic properties including chemical inertness, thermal stability, and good biocompatibility. The present work reports the development of a bio-sensing methodology which involves the immobilization of horseradish peroxidase enzyme (HRP) on pencil graphite electrode (PGE) using TiO2 sol-gel and its application for 5-aminosalicylic acid (5-ASA) determination in pharmaceutical formulations. TiO2 sol-gel film was deposited on the PGE surface by dip coating, and the HRP enzyme was immobilized on the film by adsorption and cross-linked binding using glutaraldehyde. The analytical performance of the methodology was investigated through amperometric measurements at -0.08 V in the presence of 5-ASA (electron mediator) and hydrogen peroxide (substrate). The biosensor was successfully applied for 5-ASA determination, where limits of detection and quantification of 3.3 and 10.0 µmol L -1, respectively, were obtained. The application of the methodology for 5-ASA determination in pharmaceutical formulations led to satisfactory recovery rates ranging from 80 to 98%. The biosensor developed in this work is simple, inexpensive and has the potential to be applied for the determination of other pharmaceutical compounds, which also act as electron mediators in the catalytic cycle of HRP.

Eletrodo de diamante foi usado para avaliar a interação dos nucleotídeos guanosina monofosfato (GMP) e adenosina monofosfato (AMP) com os pesticidas clorpirifós, metamidofós e monocrotofós. Observaram-se alterações nas correntes e potenciais de picos dos voltamogramas dos nucleotídeos na presença dos pesticidas dependendo da concentração de pesticida (de 5,0 × 10-7 a 5,0 × 10-5 mol L-1) e do tempo de interação (de 1 min a 4 h). Isto é provavelmente devido à ligação dos pesticidas às bases nitrogenadas presentes nos nucleotídeos, o que poderia levar a problemas na replicação do DNA e nas funções biológicas de nucleotídeos. Os pesticidas apresentaram maior interação com AMP que com GMP. Estudos sobre a interação de DNA 50 µg mL-1 com os pesticidas (de 30 min até 4 h, e de 1,0 × 10-6 a 6,0 × 10-5 mol L-1) não revelaram picos relativos à abertura da dupla hélice ou desenrolamento do DNA.

Diamond electrode was used to evaluate the interaction of the nucleotides guanosine monophosphate (GMP) and adenosine monophosphate (AMP) with the pesticides chlorpyrifos, methamidophos and monocrotophos. Changes were observed in the currents and peak potentials of the nucleotide voltammograms in the presence of the pesticides, with dependence on the pesticide concentration (from 5.0 × 10-7 to 5.0 × 10-5 mol L-1) and the interaction time (from 1 min to 4 h). This is probably due to binding of the pesticides to the nitrogenous bases present in the nucleotides, which could lead to problems in the DNA replication and biological functions of nucleotides. The pesticides showed stronger interaction with AMP than with GMP. Studies of the interaction of 50 µg mL-1 DNA with the pesticides (from 30 min to 4 h and from 1.0 × 10-6 to 6.0 × 10-5 mol L-1) did not reveal any peaks relating to double helix opening or DNA unwinding.

A espectrofotometria UV-Vis foi utilizada para medir a solubilidade do corante disperso red 1 (DR1) em soluções aquosas, com diferentes solventes e dispersantes. O corante foi quantificado em amostras de águas, e sua interação com o ds-DNA de timo de vitelo foi investigada. Os resultados mostraram que o dispersante comercial Fongranal® FB foi adequado para a preparação das soluções aquosas do DR1. Para a determinação de DR1 em água pura, o limite de detecção foi de 2,47 × 10-6 mol L-1, e o limite de quantificação, 8,22 × 10-6 mol L-1. Porcentagens de recuperação de 91,2 e 103% foram obtidas para as concentrações de DR1 em 3,00 × 10-6 e 40,0 × 10-6 mol L-1, respectivamente. As recuperações alcançadas de DR1 em água de torneira e de rio ficaram na faixa de 85,9-113%. A interação de DR1 com o DNA de timo de vitela foi acompanhada por efeitos de hipocromismo e hipercromismo, que foram relacionados à mudanças conformacionais e lesão na dupla hélice do DNA.

UV-Vis spectrophotometry was used to measure the solubility of disperse red 1 (DR1) dye in aqueous solutions, using different solvents and dispersants. The dye was quantified in water samples, and its interaction with calf thymus ds-DNA was investigated. The results showed that the commercial dispersant Fongranal® FB was suitable for the preparation of aqueous DR1 solutions. For the determination of DR1 in purified water, the limit of detection was 2.47 × 10-6 mol L-1, and the limit of quantification was 8.22 × 10-6 mol L-1. Percentage recoveries of 91.2 and 103% were obtained for DR1 concentrations of 3.00 × 10-6 and 40.0 × 10-6 mol L-1, respectively. The recoveries achieved for DR1 present in tap and river water were in the range 85.9-113%. The interaction of DR1 with calf thymus DNA was accompanied by hypochromic and hyperchromic effects, which were related to conformational changes and damage to the DNA double helix.

A detecção individual e em mistura equimolar dos nucleotídeos de DNA guanosina (GMP), adenosina (AMP), timidina (TMP) e citidina (CMP) 5'-monofosfato, utilizando a voltametria de onda quadrada, foi realizada sobre o eletrodo de diamante dopado com boro (DDB) prétratado catodicamente (Red-DDB) e anodicamente (Oxi-DDB). A oxidação dos nucleotídeos individuais de DNA foi mais sensível sobre o Oxi-DDB. Na detecção simultânea de nucleotídeos, as respostas de GMP, AMP, TMP e CMP foram muito adequadas sobre ambos os eletrodos tratados. Particularmente, picos mais sensíveis e mais separados para TMP e CMP sobre o Oxi-DDB e Red-DDB, respectivamente, foram observados após procedimento de deconvolução. A detecção dos nucleotídeos em soluções aquosas certamente contribuirá para avaliação genotóxica de substâncias e em reações de hibridização por meio da imobilização de ss ou ds-DNA sobre a superfície do DDB.

The individual detection and equimolar mixture of DNA nucleotides guanosine monophosphate (GMP), adenosine monophosphate (AMP), thymidine (TMP) and cytidine (CMP) 5'-monophosphate using square wave voltammetry was performed on boron doped diamond (BDD) electrodes cathodically (Red-DDB) and anodically (Oxi-DDB) pretreated. The oxidation of individual DNA nucleotides was more sensitive on Oxi-BDD electrode. In a simultaneous detection of nucleotides, the responses of GMP, AMP, TMP and CMP were very adequate on both treated electrodes. Particularly, more sensitive and separate peaks for TMP and CMP on Oxi-BDD and Red-BDD electrodes, respectively, were observed after deconvolution procedure. The detection of nucleotides in aqueous solutions will certainly contribute for genotoxic evaluation of substances and hybridization reactions by immobilizing ss or ds-DNA on BDD surface.

An analytical method for the determination of the anti-inflammatory drug 5-aminosalicylic acid (5-ASA) in pharmaceutical formulations using square wave voltammetry at pencil graphite electrodes was developed. After the optimization of the experimental conditions, calibration curves were obtained in the linear concentration range from 9.78 × 10-7 to 7.25 × 10-5 mol L-1 resulting in a limit of detection of 2.12 ± 0.05 x 10-8 mol L-1. Statistical tests showed that the concentrations of 5-ASA in commercial tablets and enemas obtained with the proposed voltammetric method agreed with HPLC values at a 95% confidence level.

O planejamento fatorial fracionário e com ponto central foram aplicados no desenvolvimento de um biossensor amperométrico para determinação do vírus da hepatite C. As biomoléculas foram imobilizadas por adsorção em eletrodos de grafite modificados com filme sol-gel preparado com matriz híbrida siloxano-poli(propileno-óxido). Diversos parâmetros foram otimizados, tais como a concentração da estreptavidina em 0,01mg mL-1 e albumina de soro bovino em 1,0%, o tempo de incubação dos eletrodos de 30 minutos na solução do DNA complementar e a diluição do conjugado avidina-peroxidase em 1:1500, entre outros. A aplicação de estudos quimiométricos mostrou-se eficiente, pois as melhores condições foram estabelecidas com um menor número de experimentos, além de apontar quais os fatores exercem maiores influências sobre o sistema.

Fractional factorial design and factorial with center point design were applied to the development of an amperometric biosensor for the detection of the hepatitis C virus. Biomolecules were immobilized by adsorption on graphite electrodes modified with siloxane-poly(propyleneoxide) hybrid matrix prepared using the sol-gel method. Several parameters were optimized, such as the streptavidin concentration at 0.01 mg mL-1 and 1.0% bovine serum albumin, the incubation time of the electrodes in the complementary DNA solution for 30 minutes and a 1:1500 dilution of the avidin-peroxidase conjugate, among others. The application of chemometric studies has been efficient, since the best conditions have been established with a restricted number of experiments, indicating the influence of different factors on the system.