RESUMO
The design and characterization of a new nanostructured organic-inorganic hybrid material and its application to L-lactic acid determination are described. This material is based on the integration of the enzyme lactate oxidase (LOx) and gold nanoparticles (AuNPs) into a sol-gel 3D polymeric network derived from (3-mercaptopropyl)-trimethoxysilane (MPTS) previously formed onto a gold surface. MPTS presents the advantage of forming a 3D polymeric network containing a large number of thiol tail groups distributed throughout its structure that enable both its anchoring onto gold surfaces and the AuNPs incorporation. Moreover, this matrix provides a biocompatible environment that preserves the catalytic activity of LOx after its immobilization and allows the incorporation of a high amount of enzyme, which is expected to improve the sensitivity of the final biosensing device. Characterization of the designed biosensing platform was performed using quartz crystal microbalance (QCM), scanning electron microscopy (SEM) and atomic force microscopy (AFM) techniques. From the conjunction of these techniques, information about (i) the kinetic of LOx adsorption process in real time, (ii) the amount of LOx incorporated into the network, and (iii) the morphological characteristics at the nanometre level of the designed biosensing material was obtained. This information is very useful on the development of successful biosensing devices. Finally, the response of the biosensor to L-lactic acid was evaluated. The biosensor responds linearly to L-lactic acid in the range of 50 µM to 0.25 mM, with a sensitivity of 3.4 µA mM(-1) and a detection limit of 4.0 µM.
Assuntos
Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Ácido Láctico/análise , Oxigenases de Função Mista/metabolismo , Nanoestruturas/química , Pediococcus/enzimologia , Silanos/química , Enzimas Imobilizadas/metabolismo , Ouro/química , Ácido Láctico/metabolismo , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Nanoestruturas/ultraestrutura , Compostos de Organossilício , Sensibilidade e EspecificidadeRESUMO
The design and characterization of a new organic-inorganic hybrid composite material for glucose electrochemical sensing are described. This material is based on the entrapment of both gold nanoparticles (AuNPs) and glucose oxidase, which was chosen as a model, into a sol-gel matrix. The addition of spectroscopic grade graphite to this system, which confers conductivity, leads to the development of a material particularly attractive for electrochemical biosensor fabrication. The characterization of the hybrid composite material was performed using atomic force microscopy and scanning electron microscopy techniques. This composite material was applied to the determination of glucose in presence of hydroxymethylferrocene as a redox mediator. The system exhibits a clear electrocatalytic activity towards glucose, allowing its determination at 250 mV vs Ag/AgCl. The performance of the resulting enzyme biosensor was evaluated in terms of sensitivity, detection limit, linear response range, stability and accuracy. Finally, the enhancement of the analytical response of the resulting biosensor induced by the presence of gold nanoparticles was evaluated by comparison with a similar organic-inorganic hybrid composite material without AuNPs.
Assuntos
Técnicas Biossensoriais/métodos , Eletroquímica/métodos , Ouro/química , Compostos Inorgânicos/química , Nanopartículas Metálicas/química , Compostos Orgânicos/química , Técnicas Biossensoriais/instrumentação , Eletroquímica/instrumentação , Eletrodos , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Géis/química , Glucose/química , Glucose Oxidase/química , Glucose Oxidase/metabolismo , Grafite/química , Nanopartículas Metálicas/ultraestrutura , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Polimetil Metacrilato/química , Reprodutibilidade dos Testes , Propriedades de SuperfícieRESUMO
Forty-three faecal non-enterotoxigenic and non-enteropathogenic human Escherichia coli strains causing mannose-resistant haemagglutination (MRHA) were tested for production of cytotoxic necrotizing factor (CNF), haemolysis (Hly), Verotoxin (VT) and lethal activity for mice. The serotypes of the strains were also determined. Of the total strains investigated, 49% synthesized CNF, 53% were haemolytic and 40% were lethal for mice. No strain producing VT was detected. Striking differences in the production of Hly and CNF were observed when MRHA strains were grouped according to their lethal or non-lethal activity. Thus, 82% of lethal strains produced Hly and/or CNF whereas only 35% (p less than 0.01) and 27% (p less than 0.01) of non-lethal strains produced Hly and CNF, respectively. The production of toxins was specially associated with strains possessing defined MRHA types. Thus, 100%, 82% and 50% of strains belonging to MRHA types III, IVa and V, respectively, were toxigenic, whereas no toxigenic strains from MRHA types IVb and VI were detected. The majority (77%) of MRHA strains possessed typical O groups usually reported to be present in pathogenic extraintestinal E. coli or in facultatively enteropathogenic E. coli. Furthermore, these O groups were more frequently detected in toxigenic (93%) than in non-toxigenic (47%) strains (p less than 0.01). Our results suggest that faecal non-enterotoxigenic E. coli strains belonging to MRHA types III, IVa and V may be responsible for extraintestinal infections as well as for sporadic intestinal infections, and that certain O groups are specially associated with E. coli strains belonging to particular MRHA types.
