Assuntos
Anacardium/química , Anti-Infecciosos/química , Antineoplásicos/química , Cobre/química , Nanopartículas Metálicas/química , Gomas Vegetais/química , Animais , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Sobrevivência Celular , Sulfato de Cobre/química , Sulfato de Cobre/toxicidade , Eritrócitos/citologia , Macrófagos/citologia , Nanopartículas Metálicas/toxicidade , Camundongos , Camundongos Endogâmicos BALB C , Ovinos , Staphylococcus aureus/efeitos dos fármacos , Propriedades de Superfície , Fatores de TempoRESUMO
A new selective electrochemical genosensor has been developed for the detection of an 86-mer DNA peanut sequence encoding part of the allergen Ara h 2 (conglutin-homolog protein). The method is based on a sandwich format, which presents two advantages: it permits shortening the capture probe and avoids labeling of the target. Screen-printed gold electrodes have been used as platform for the immobilization of oligonucleotides by the well-known S-Au bond. Mixed self-assembled monolayers (SAM), including thiol-modified capture probe and mercaptohexanol, were prepared to achieve an organized, homogeneous and not too compact SAM in which unspecific adsorption of the capture probe would be prevented. The optimization of the sensing phase was carried out using the Design of Experiments (DoE) approach. Traditionally, response optimization is achieved by changing the value of one factor at a time until there is no further improvement. However, DoE involves regulating the important factors so that the result becomes optimal. Optimized conditions were found to be 1.34 µM for capture probe concentration and 3.15 mM for mercaptohexanol (spacer) concentration. When the optimal conditions were employed the analytical performance of the proposed genosensor improved significantly, showing a sensitivity as high as 3 µA/nM, with a linear range from 5×10(-11) to 5×10(-8) M and a detection limit of 10 pM.
Assuntos
Albuminas 2S de Plantas/análise , Alérgenos/análise , Antígenos de Plantas/análise , Técnicas Biossensoriais/métodos , Glicoproteínas/análise , Albuminas 2S de Plantas/genética , Alérgenos/genética , Antígenos de Plantas/genética , Arachis/efeitos adversos , Arachis/genética , Arachis/imunologia , Sequência de Bases , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/estatística & dados numéricos , Calibragem , Sondas de DNA/química , Sondas de DNA/genética , DNA de Plantas/química , DNA de Plantas/genética , Técnicas Eletroquímicas , Análise de Alimentos , Glicoproteínas/genética , Humanos , Limite de Detecção , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Reprodutibilidade dos TestesRESUMO
This paper proposes a new concept of transketolase (TK) activity profiling. A tyrosinase (PPO) biosensor, based on the immobilization of this enzyme in a Mg(2)Al-Cl layered double hydroxide, was developed for the amperometric detection of N-acetyl-l-tyrosine ethyl ester monohydrate (N-Ac-Tyr-OEt) at -0.2V. This compound was released during an enzymatic reaction catalyzed by TK with N-acetyl-O-(2R, 3S, 5-trihydroxy-4-oxopentyl)-l-tyrosine ethyl ester used as donor substrate. This tyrosinase biosensor was optimized for the detection of TK activity, including PPO optimum substrate concentration, electrolyte nature, pH, and influence of bovine serum albumin (BSA). It was found that N-Ac-Tyr-OEt release is dependent on TK concentration (U/mL) in the electrolyte medium. These results demonstrate the sensitivity and specificity of the tyrosinase biosensor designed for in vitro detection of TK activity, which is known to be involved in several diseases.
